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Originally published In Press as doi:10.1074/jbc.M402095200 on March 29, 2004

J. Biol. Chem., Vol. 279, Issue 22, 23207-23213, May 28, 2004
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Peroxiredoxin-null Yeast Cells Are Hypersensitive to Oxidative Stress and Are Genomically Unstable*

Chi-Ming Wong, Kam-Leung Siu, and Dong-Yan Jin, Leukemia and Lymphoma Society Scholar{ddagger}

From the Department of Biochemistry, University of Hong Kong, Hong Kong, China

Received for publication, February 25, 2004 , and in revised form, March 26, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Peroxiredoxins are a family of abundant peroxidases found in all organisms. Although these antioxidant enzymes are thought to be critically involved in cellular defense and redox signaling, their exact physiological roles are largely unknown. In this study, we took a genetic approach to address the functions of peroxiredoxins in budding yeast. We generated and characterized a yeast mutant lacking all five peroxiredoxins. The quintuple peroxiredoxin-null mutant was still viable, though the growth rate was lower under normal aerobic conditions. Although peroxiredoxins are not essential for cell viability, peroxiredoxin-null yeast cells were more susceptible to oxidative and nitrosative stress. In the complete absence of peroxiredoxins, the expression of other antioxidant proteins including glutathione peroxidase and glutathione reductase was induced. In addition, the quintuple mutant was hypersensitive to glutathione depletion. Thus, the glutathione system might cooperate with other antioxidant enzymes to compensate for peroxiredoxin deficiency. Interestingly, the peroxiredoxinnull yeast cells displayed an increased rate of spontaneous mutations that conferred resistance to canavanine. This mutator phenotype was rescued by yeast peroxiredoxin Tsa1p, but not by its active-site mutant defective for peroxidase activity. Our findings suggest that the antioxidant function of peroxiredoxins is important for maintaining genome stability in eukaryotic cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Peroxiredoxins are a family of thiol-specific peroxidases found across all kingdoms of living organisms (1-4). All peroxiredoxins conserve at least one cysteine motif at their active center. Many of them are known to form oligomers (5, 6). Peroxiredoxins can reduce a wide range of peroxides. Their peroxidase (7, 8) and peroxynitrite reductase (9-11) activities depend on the thiol-disulfide transition of cysteines supported by electron donors such as thioredoxin and cyclophilin (12, 13). In addition, peroxiredoxins have also been suggested to regulate redox signaling (1, 14, 15), likely through the reversible oxidation of cysteine to cysteine sulfinic acid (16, 17). A recent study has demonstrated that the reduction of cysteine sulfinic acid requires ATP hydrolysis and is catalyzed by sulfiredoxin, which forms mixed disulfide intermediate with peroxiredoxins (18).

Multiple peroxiredoxins are commonly found in one species. Thus, there are three peroxiredoxins in Escherichia coli (19), five in Saccharomyces cerevisiae (20), and at least six in mammals (21, 22). Four of the five peroxiredoxins in budding yeast have human orthologs. Thus, the yeast serves as a good model for studying the biological functions of peroxiredoxins. Yeast peroxiredoxins are variously termed as Tsa1p/cTPxI/YML028W (7, 20), Tsa2p/cTPxII/YDR453C (11, 20), Dot5p/nTPx/BCP/YIL010W (23, 24), Prx1p/mTPx/1CPrx/YBL064C (25), and Ahp1p/cTPxIII/PMP20/YLR109W (26). All five enzymes have similar antioxidant properties, but are differentially expressed and localized (20).

Construction of null mutants in budding yeast is a powerful means to understanding the physiology of peroxiredoxins. Single peroxiredoxin-null mutants have indicated that none of the five peroxiredoxins in yeast is essential (20). However, a tsa1{Delta} tsa2{Delta} double mutant is much more sensitive to oxidative and nitrosative stress, suggesting that different peroxiredoxins might cooperate with each other in the antioxidant defense (11). Several lines of additional evidence lend further support to the notion that different peroxiredoxins might serve overlapping functions in the yeast cell. First, all yeast peroxiredoxins have thioredoxin peroxidase activity (7, 8, 20, 25). Second, although peroxiredoxins are found in different compartments of the cell (20), several of them are also expressed in the cell at the same time and place (3, 27, 28). Third, the expression of peroxiredoxin genes is co-regulated through similar stress response pathways and similar transcription factors (23, 29-31). Finally, when one peroxiredoxin is compromised, compensational activation of other peroxiredoxins has been observed (11).

To challenge the possibility that peroxiredoxin-null single or double mutants are viable because of functional overlaps between different peroxiredoxins, we constructed yeast mutants lacking multiple peroxiredoxins, including a quintuple mutant in which all peroxiredoxin genes were disrupted. Remarkably, all peroxiredoxin-null mutants were viable. The complete ablation of yeast peroxiredoxin genes induced compensational expression of other antioxidant proteins. In particular, the quintuple mutants were more sensitive to glutathione depletion. Notably, the gene mutability of peroxiredoxin-null mutants was found to increase significantly. In addition, the expression of Tsa1p but not its active-site mutant complemented this mutator phenotype. Our findings implicate a role of peroxiredoxins in maintaining genome stability.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Yeast Strains and Media—S. cerevisiae strain BY4741 (32) and its isogenic strains (Table I) were used. Media were prepared and cell transformation was performed as described (11, 31).


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  		TABLE I
Yeast strains

 
Mutant Construction—Peroxiredoxin-null mutants were constructed with a one-step gene disruption method (33) and/or PCR-based gene deletion strategy (34). Details of primers and plasmids are available upon request.

Construction of TSA1 Expression Plasmids—The TSA1 expression plasmid has been described elsewhere (11). Site-directed mutagenesis was performed to replace cysteines 47 and 170 of Tsa1p with serines. DNA sequencing confirmed that all mutations were successfully introduced.

RT-PCR—RT-PCR was performed as described (11). The PCR conditions were optimized to ensure that the amplification was in the linear range.

Detection of Reactive Oxygen and Nitrogen Species—Intracellular levels of reactive oxygen species (ROS)1 were measured by fluorimetry using the fluorescent dye 2',7'-dichlorofluorescein diacetate (DCF-DA; Molecular Probes) as described (11). The intracellular levels of reactive nitrogen species (RNS) were measured by the same method using another probe, 4',5'-diaminofluorescein diacetate (DAF-DA; Molecular Probes). DAF fluorescence was measured with F-4500 spectrofluorimeter (Hitachi). Spot assays for sensitivity to oxidants were performed as described (11).

Determination of Free Glutathione Levels—Glutathione levels were determined by using 5',5'-dithiobis-(2)-nitrobenzoic acid (DTNB; Calbiochem) as described previously (35). In brief, cells were grown in GSH-free S.D. medium until A600 reached 1. After treatment with H2O2, sodium nitroprusside (SNP), or 1-chloro-2,4-dinitrobenzene (CDNB; Calbiochem), cells were harvested by centrifugation and washed three times with phosphate-buffered saline, pH 7.4. Cells were resuspended in ice-cold 8 mM HCl and 1.3% (w/v) 5-sulfosalicyclic acid and then disrupted with glass beads. The cell debris was pelleted by centrifugation. The supernatant was neutralized with 100 mM potassium phosphate, pH 7.4, and the level of free glutathione was determined. DTNB was added to a final concentration of 0.4 mg/ml, and incubated in dark at room temperature for 15 min. The absorbance was measured at 414 nm.

Fluctuation Analysis—The number of cells mutated in the population was determined by fluctuation analysis (36). In brief, cells were grown to saturation, diluted to different concentrations, and spread on yeast extract/peptone/dextrose (YPD) plate with canavanine (60 mg/liter; Sigma). The number of colonies with canavanine-resistant (Canr) cells was calculated. At least five independent cultures were analyzed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of Quintuple Peroxiredoxin-null Mutant—We constructed the tsa1{Delta} tsa2{Delta} prx1{Delta} dot5{Delta} ahp1{Delta} (prx{Delta}) strain by replacing the TSA1 coding region with KAN, TSA2 with LEU2, PRX1 with URA3, DOT5 with MET15, and AHP1 with HIS3. Genomic PCR (Fig. 1A) was performed to confirm the insertion of the markers into the peroxiredoxin genes. In addition, no TSA1, TSA2, PRX1, DOT5, or AHP1 mRNA was detected in prx{Delta} cells by RT-PCR (Fig. 1B). Both experiments consistently verified the disruption of all peroxiredoxin genes in the prx{Delta} strain.



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  		FIG. 1.
Disruption of PRX1, TSA1, TSA2, DOT5, and AHP1 in prx{Delta} strain. A, genomic PCR analysis. Disrupted and intact PRX1, TSA1, TSA2, DOT5, and AHP1 genes in the prx{Delta} (5{Delta}; lanes 2, 4, 6, 8, and 10) and BY4741 isogenic wild-type (WT; lanes 1, 3, 5, 7, and 9) yeast strains were detected by PCR with specific primers. The expected sizes of the PCR product for intact PRX1, TSA1, TSA2, DOT5, and AHP1 were 875, 869, 666, 887, and 949 bp, respectively. M, DNA molecular size marker. B, RT-PCR analysis. RT-PCR was performed to verify the absence of peroxiredoxin transcripts in prx{Delta} strain. Primers bound to the removed regions were used. The housekeeping gene ORC5 was used as a positive control. The expected sizes of the PCR product for intact PRX1, TSA1/TSA2, DOT5, and AHP1 are 327, 247, 278, 251, and 284 bp, respectively. The primer-binding sequences in TSA1 and TSA2 are identical.

 
The prx{Delta} Mutant Is Viable—The prx{Delta} cells were viable in aerobic culture and they grew normally as compared with the isogenic wild-type BY4741. We noted that the growth rate of prx{Delta} cells was lower than that of BY4741 during late log and stationary phases (Fig. 2A). This phenotype was probably ascribed to the loss of TSA1, which does not affect growth during early log phase (11) but is associated with a late onset of slow growth (37-39). In this regard, the tsa2{Delta}, prx1{Delta}, dot5{Delta}, and ahp1{Delta} single mutants do not grow significantly slower in any phase under standard aerobic conditions (11, 20). We also compared the cell cycle profiles of BY4741 and prx{Delta} cells by flow cytometric analysis of DNA content (Fig. 2B). The distribution of G1/S and G2/M cells in the two strains was found to be similar, suggesting that the prx{Delta} cells did not suffer drastic changes in cell cycle progression. Thus, yeast peroxiredoxins are dispensable to cell survival.



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  		FIG. 2.
Growth of prx{Delta} cells in aerobic condition. A, growth rate. Wild-type BY4741 ({blacksquare}) and prx{Delta} ({square}) strains were grown aerobically in YPD medium at 30 °C for 36 h. B, flow cytometric analysis of DNA content. Ten thousand midlog-phased cells were analyzed. The two peaks represent 1N and 2N DNA content, corresponding to G1/S and G2/M cells. Similar results were obtained from three independent experiments.

 
The prx{Delta} Mutant Is Hypersensitive to ROS and RNS—Yeast peroxiredoxins have thioredoxin peroxidase and peroxynitrite reductase activities in vitro (11, 20). In addition, single and double peroxiredoxin-null mutants are sensitive to ROS and RNS (11, 20). To address whether different peroxiredoxins might have overlapping functions in the cellular defense against oxidative and nitrosative stress, we challenged the peroxiredoxin-null single, double, triple, quadruple, and quintuple mutants with various oxidants. Spot assays were performed to determine the tolerance of the cells to the challenge (Fig. 3A). All peroxiredoxin-null mutants, including prx{Delta}, had no dramatic change in growth rate under normal aerobic condition on YPD agar at 30 °C (Fig. 2A and data not shown). However, to different degrees, these mutants were hypersensitive to ROS and RNS induced by hydrogen peroxide (H2O2), t-butyl-hydroperoxide (tBHP), peroxynitrite (PN), and SNP. In general, the mutant cells defective for multiple peroxiredoxin genes were more susceptible to ROS and RNS. Accordingly, the prx{Delta} cells were most sensitive to all insults. These results are consistent with the concept that different peroxiredoxins might cooperate with each other in the antioxidant defense.



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  		FIG. 3.
Peroxiredoxin-null mutants are hypersensitive to oxidants and have higher levels of intracellular ROS and RNS. A, spot assay. The indicated cells were grown in YPD medium until A600 reached 1 and were then treated for 30 min with 4 mM hydrogen peroxide (H2O2), 1.5 mM t-butyl-hydroperoxide (tBHP), 2 mM peroxynitrite (PN), 2 mM sodium nitroprusside (SNP), 0.5 mM nystatin, and 0.2 µg/ml antimycin A (AMA), respectively. Serial dilutions (0, undiluted; -2, diluted 102-fold; and -4, diluted 104-fold) of cells were spotted on YPD plates. The cells were incubated at 30 °C for 2 days. B, ROS detection. The wild-type and mutant cells were grown in YPD medium until A600 reached 1, either mock-treated ({square}) or treated with 0.5 mM H2O2 ({blacksquare}), and incubated for 15 min. DCF-DA was then added to 10 µM, and the cells were further incubated at 30 °C for 30 min. The cells were disrupted, and the crude extracts containing 500 µg of protein were suspended in phosphate-buffered saline. DCF fluorescence was measured on an F-4500 spectrofluorimeter (Hitachi). The excitation and emission wavelengths were 488 and 520 nm, respectively. The fluorescent intensity of the BY4741 cells was taken as 100%. C, RNS detection. The assay was performed as described above, except that the cells were treated with 1 mM SNP ({blacksquare}) for 15 min, and DAF-DA was used as a probe for RNS. The excitation and emission wavelengths were 495 and 515 nm, respectively.

 
The mitochondrial respiratory chain is a major source of intracellular ROS and RNS. To cope with this, the cell has developed mitochondrial antioxidant systems (40). Although yeast peroxiredoxin Tsa1p in the cytoplasm has previously been implicated in the cellular response to the inhibition of mitochondrial function (41), the mitochondrial peroxiredoxin Prx1p (25) could be a more physiologically relevant antioxidant enzyme for scavenging ROS and RNS generated from respiration. To address this issue, we treated various yeast mutants separately with nystatin and antimycin A (AMA), which disrupt mitochondrial electron transport. To our surprise, most of the peroxiredoxin-null mutants including the prx1{Delta} strain were only slightly more sensitive to nystatin or AMA (Fig. 3A). In contrast, prx{Delta} and one quadruple mutant (4{Delta}) exhibited a moderate susceptibility to nystatin or AMA (Fig. 3A). Generally, the mutants were more susceptible when multiple peroxiredoxin genes were disrupted. These data are also compatible with the model that different peroxiredoxins might functionally complement each other.

Elevated Intracellular ROS and RNS Levels in prx{Delta} Cells— Because peroxiredoxins are ROS and RNS scavengers, the complete loss of peroxiredoxins would plausibly disrupt redox homeostasis in cells. To measure intracellular ROS and RNS levels in the prx{Delta} mutant cells, we used DCF-DA and DAF-DA dyes. These are cell-permeable fluorogenic probes that have been widely used for ROS and RNS detection (11, 42-45). The relative DCF and DAF fluorescence intensities can reflect the levels of overall oxidative and nitrosative stress in cells.

The wild-type BY4741 and different peroxiredoxin-null mutant cells were aerobically grown in YPD medium at 30 °C, treated with DCF-DA or DAF-DA, and examined by fluorimetry. We noted that the DCF and DAF fluorescence intensities were significantly higher in peroxiredoxin-null cells than in BY4741 (Fig. 3, B and C, {square}). Thus, the loss of peroxiredoxin function plausibly led to the accumulation of ROS and RNS in cells. More interestingly, the basal levels of intracellular ROS and RNS increased progressively as more peroxiredoxin genes were disrupted (Fig. 3, B and C, compare groups 1-5). These results imply that the five peroxiredoxins function additively in scavenging ROS and RNS.

Next we challenged the yeast cells with hydrogen peroxide and SNP before measuring the ROS and RNS levels. As expected, the addition of hydrogen peroxide and SNP further increased intracellular ROS and RNS over the basal level (Fig. 3, B and C, {blacksquare}). However, the relative ROS and RNS levels in different strains showed the same pattern. Consistently, progressively more ROS and RNS were detected in the BY4741, double (2{Delta}), triple (3{Delta}), quadruple (4{Delta}), and quintuple (5{Delta}) mutants (Fig. 3, B and C, compare groups 1-5), indicating that the loss of each peroxiredoxin gene contributes to the accumulation of intracellular ROS and RNS.

Induced Expression of Other Antioxidant Enzymes in prx{Delta} Cells—As shown above, the intracellular ROS and RNS levels in prx{Delta} cells were elevated (Fig. 3), but the prx{Delta} cells did not exhibit serious growth defects in aerobic culture (Fig. 2). We postulated that the prx{Delta} yeast cells might have adapted to the higher intracellular ROS and RNS levels by overexpressing other antioxidant enzymes, likely through the activation of redox-regulated transcription factors such as Msn2p, Msn4p, and Yap1p (29, 46, 47). Such adaptive response may protect the cells against excess ROS and RNS.

To test our hypothesis, we used semi-quantitative RT-PCR to assess the mRNA expression of a number of antioxidant enzymes including catalase 1 (Ctt1p), Cu, Zn superoxide dismutase 1 (Sod1p), thioredoxin reductase 1 (Trr1p), thioredoxin 1 (Trx1p), thioredoxin 2 (Trx2p), glutathione peroxidase 2 (Gpx2p), and glutathione reductase 1 (Glr1p). Indeed, the steady-state amounts of the transcripts of these antioxidant proteins were induced in prx{Delta} (5{Delta}) cells (Fig. 4). Compared with the amounts in wild-type BY4741 cells, the levels of SOD1, TRX2, and GLR1 transcripts rose remarkably in prx{Delta} cells after challenging with H2O2 (Fig. 4, compare lane 4 to lane 3). CTT1, TRX1, and GPX2 transcripts were induced in prx{Delta} cells even in the absence of H2O2 (Fig. 4, compare lane 2 to lane 1). The induction of these antioxidant enzymes might compensate for peroxiredoxin deficiency. Therefore, peroxiredoxins not only cooperate with each other, but they also cooperate with other antioxidant defense systems in S. cerevisiae.



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  		FIG. 4.
RT-PCR analysis of the mRNA transcripts of some antioxidant enzymes in prx{Delta} cells. BY4741 (WT) and prx{Delta} (5{Delta}) cells were mock-treated (lanes 1 and 2) or treated with 0.5 mM H2O2 (lanes 3 and 4). The housekeeping gene ORC5 was used as a positive control.

 
The prx{Delta} Cells Have Higher Levels of Reduced Glutathione and Are Hypersensitive to Glutathione Depletion—Glutathione plays an important role in maintaining the redox balance in cells. The glutathione system is also thought to cooperate with the thioredoxin system in cellular antioxidant defense (48). We have documented the compensational expression of glutathione reductase and glutathione peroxidase in prx{Delta} cells (Fig. 4). To follow up, we measured the levels of the reduced glutathione (GSH) in the peroxiredoxin-null mutants by DTNB (35). The GSH levels in the peroxiredoxin-null mutants were higher than in BY4741 (Fig. 5A, {square}). Again, the loss of more peroxiredoxins correlated with a more significant increase in basal GSH levels. In this context, the GSH level in prx{Delta} cells was 2 times higher than in wild-type BY4741 (Fig. 5A, {square}, compare column 5 with column 1). We noted that the treatment with low-dose CDNB (0.1 µM), a common GSH-depleting agent (49), efficiently depleted GSH in all yeast strains to a similar level (Fig. 5A, {blacksquare}).



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  		FIG. 5.
Peroxiredoxin-null cells have higher levels of free GSH and are sensitive to glutathione depletion. A, GSH levels. The cells were grown in YPD medium until A600 reached 1 and were then mock-treated ({square}) or treated with 100 nM CDNB ({blacksquare}) for 1 h. The amount of GSH was measured by absorbance at 414 nm. The GSH level in BY4741 cells was taken as 100%. B-E, cellular sensitivity to glutathione depletion. BY4741 (WT) and prx{Delta} (5{Delta}) strains were grown to saturation and diluted to an A600 of 0.2. Cells were mock-treated (B) or treated with 0.5 mM H2O2 (C), 1 mM SNP (D), or 100 nM CDNB (E). The cells were then grown further in YPD medium, and A600 was measured at the indicated time points.

 
Next we asked whether GSH depletion by CDNB might affect the viability of peroxiredoxin-null mutants. Although BY4741 cells tolerated CDNB treatment well, the prx{Delta} cells in normal aerobic culture were as hypersensitive to CDNB (Fig. 5E) as to hydrogen peroxide (Fig. 5C) or SNP (Fig. 5D). Thus, GSH has a crucial protective role in the prx{Delta} cells.

The Peroxiredoxin-null Cells Are Genomically Unstable— ROS are a major cause of DNA damage in cells (50). We have demonstrated that the levels of ROS and RNS were constitutively elevated in peroxiredoxin-null cells (Fig. 3). This accumulation of ROS and RNS might plausibly lead to mutagenic alterations and genome instability. Indeed, a recent genome-wide screen of 4847 yeast gene deletions has identified TSA1 as a mutation-suppressing gene (36). The construction of multiple peroxiredoxin-null mutants has provided a new opportunity to study the roles of peroxiredoxins in the maintenance of genome stability. As a first step, we determined the mutation rates of the peroxiredoxin-null mutants. This assay was based upon the measurement of spontaneous mutations that inactivate the CAN1 gene and thereby confer resistance to canavanine. Consistent with the recent finding (36), we observed that the tsa1{Delta} strain had a higher mutation rate than the wild-type strain (Fig. 6A, compare lane 2 to lane 1). In contrast, the mutation rate of the tsa2{Delta} strain was not significantly elevated (Fig. 6A, compare lane 3 to lane 1). Likewise, the mutability of prx1{Delta}, dot5{Delta}, and ahp1{Delta} single mutants was also similar to that of the BY4741 strain (data not shown). However, the tsa1{Delta}/tsa2{Delta} double mutant (2{Delta}) had a significantly increased mutation rate than the tsa1{Delta} strain (Fig. 6A, compare lane 4 to lane 2), implicating that Tsa2p might complement Tsa1p in the suppression of gene mutations. The triple (3{Delta}), quadruple (4{Delta}), and quintuple (5{Delta}) mutants also exhibited higher mutation rates than the tsa1{Delta} single mutant (Fig. 6A, compare lanes 5-7 to lane 2), but the level was comparable with that of the double (2{Delta}) mutant (compare lanes 5-7 to lane 4). This observation could be explained by a ceiling effect.



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  		FIG. 6.
Peroxiredoxin-null mutants are hypermutable. A, mutation rates of peroxiredoxin-null mutants. The number of Canr cells was normalized with the total number of cells spread onto the plate. The mutation rate of BY4741 cells was taken as 100%. Results represent the average from three independent experiments. B, RT-PCR was performed to verify the mRNA expression of wild-type TSA1 (lane 2) and its mutant (lane 3) in tsa1{Delta} cells. C, cellular sensitivity to hydrogen peroxide. BY4741 and tsa1{Delta} cells transformed with the indicated plasmids were treated with 2 mM H2O2 for 30 min, serially diluted (0, undiluted; -2, diluted 102-fold; -4, diluted 104-fold; and -6, diluted 106-fold), and spotted onto S.D.-U (S.D. medium without uracil) plates. D, expression of Tsa1p rescues the mutator phenotype. BY4741 and tsa1{Delta} cells were transformed individually with empty pYEUra3 vector (group 1), plasmid pTSA1 expressing wild-type Tsa1p (group 2), and pTSA1CCS expressing the active-site mutant of Tsa1p (group 3), in which cysteines 47 and 170 had been replaced by serines. Transformants were selected in S.D.-U. The expression of Tsa1p was induced by 2% galactose, and the mutation was measured on S.D.-U plates with canavanine. The number of Canr cells was normalized with the total number of cells spread onto the plate. The mutation rate of BY4741 cells was taken as 100%.

 
Yeast peroxiredoxins have other functions that are independent of their antioxidant activity, including the abilities to disrupt telomeric silencing (51) or to interact with phosphatidylinositol transfer protein (52). To investigate whether the antioxidant activity of Tsa1p is important for suppressing gene mutations, we employed an active-site mutant of Tsa1p, in which cysteines 47 and 170 were substituted with serines. This mutant Tsa1pCCS has previously been shown to be defective for peroxidase activity (7) and has been widely used in functional studies (1, 2). First we performed RT-PCR analysis of mRNA transcripts and verified that both TSA1 and TSA1CCS genes were efficiently expressed in tsa1{Delta} cells (Fig. 6B). We also confirmed that the expression of Tsa1p, but not that of Tsa1pCCS, complemented the hypersensitivity of tsa1{Delta} cells to hydrogen peroxide (Fig. 6C). Finally, we observed that the expression of Tsa1p rescued the mutator phenotype of tsa1{Delta} cells (Fig. 6D, compare group 2 to group 1). In contrast, the mutation rate did not reduce when Tsa1pCCS was overexpressed (Fig. 6D, compare group 3 to group 1). Thus, the antioxidant function of Tsa1p is required for its mutation-suppressing activity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we provided the first evidence that a yeast mutant lacking all five peroxiredoxins was viable (Fig. 1 and Fig. 2). The yeast mutants defective for multiple peroxiredoxins were more susceptible to ROS/RNS insult (Fig. 3) and glutathione depletion (Fig. 5). The compensational activation of other antioxidants such as GSH and glutathione peroxidase might enable the mutant cells to survive (Fig. 4 and Fig. 5). Correlated with significantly increased levels of intracellular ROS and RNS (Fig. 3), peroxiredoxin-null yeast cells were hypermutable and genomically unstable (Fig. 6). Our findings have implications in cellular antioxidant defense and carcinogenesis.

Several lines of evidence consistently support the concept that yeast peroxiredoxins cooperate with each other to protect the cells against oxidative and nitrosative stress. First, yeast mutants lacking multiple peroxiredoxins were generally more susceptible to ROS, RNS, and inhibitors of mitochondrial respiration (Fig. 3A). Second, the levels of intracellular ROS and RNS were progressively elevated in yeast mutants defective for two, three, four, and five peroxiredoxins (Fig. 3, B and C). Third, the relative GSH levels were also higher in yeast strains lacking multiple peroxiredoxins (Fig. 5A). Finally, the tsa11{Delta}/tsa2{Delta} double mutant and other mutants in which more peroxiredoxin genes were disrupted had higher mutation rates than the single mutants (Fig. 6A). However, considered together with previous findings (11, 20), our data also suggest that the five peroxiredoxins might have distinct and non-overlapping functions. As such, Tsa1p has a more dominant role in antioxidant defense among the five peroxiredoxins. Compared with the other single mutants, the tsa1{Delta} strain exhibited the most dramatic phenotype in sensitivity (11) and mutability (Fig. 6A) assays. The other peroxiredoxins became more important when TSA1 is lost (Figs. 3A, 5A, and 6A).

Peroxiredoxins are important in antioxidant defense. Loss of peroxiredoxins led to the accumulation of ROS and RNS in cells (Fig. 3). In line with previous findings (53), we observed an induction of other antioxidant enzymes in peroxiredoxin-null cells (Fig. 4). This induction probably enables the cells to survive the complete loss of peroxiredoxins. Thus, peroxiredoxins cooperate not only with each other, but also with other antioxidants in the defense against ROS and RNS. We showed that the levels of GSH, Gpx2p, and Glr1p are elevated in peroxiredoxin-null cells (Fig. 4 and Fig. 5A). In addition, the quintuple mutant is particularly sensitive to glutathione depletion (Fig. 5E), implicating a crucial role of the glutathione system in compensating for peroxiredoxin deficiency. Our data are complementary to the previous finding that overexpression of peroxiredoxin protects the cells from glutathione depletion (54). In this regard, the functional overlap between the thioredoxin and glutathione systems has been well documented (48, 55, 56). It is also noteworthy that peroxiredoxins and glutathione peroxidases share the same properties, including the use of sulfhydryl groups to detoxify peroxides, the formation of similar reaction intermediates, and the ability to reduce a wide spectrum of substrates such as H2O2, lipid hydroperoxides, and peroxynitrite (9, 11, 55, 57). Thus, it is not surprising that peroxiredoxins and glutathione peroxidases may complement each other in detoxifying peroxides and protecting the cells from oxidative stress.

Our study on genome instability of peroxiredoxin-null strains (Fig. 6) corroborates the recent finding from a genomewide screen that peroxiredoxins have a role in the suppression of gene mutations (36). We found that the mutation rates of peroxiredoxin-null strains were further increased when more peroxiredoxin genes were knocked out (Fig. 6A). Notably, this increase in gene mutability correlated with the elevation of intracellular ROS and RNS levels (Fig. 3). Using an active-site mutant of Tsa1p, we confirmed that the peroxidase activity of peroxiredoxins is important for the suppression of mutations. Plausibly, the ability of peroxiredoxins to suppress gene mutations is attributed to the removal or detoxification of excessive ROS and RNS that are known to cause DNA damage (50, 58, 59).

Hypermutability and genome instability are hallmarks of cancer. Interestingly, a recent study has demonstrated that mice lacking Prdx1, an ortholog of Tsa1p, develop hemolytic anemia and malignant cancers including lymphomas, sarcomas, and carcinomas (60). These results provide the first direct evidence for a role of peroxiredoxins in carcinogenesis. The Prdx1-deficient mice and the mice lacking either Prdx2 or Prdx6 are also hypersensitive to oxidative stress and DNA damage (60-62). These observations, together with our study, implicate peroxiredoxins as a novel group of tumor-suppressor proteins that have protective roles against DNA damage and genome instability caused by free radicals. Further investigations are required to elucidate the particular roles and mechanisms of peroxiredoxins in genome maintenance and tumor suppression.


   FOOTNOTES
 
* This work was supported by Project Grants HKU 7240/00M and HKU 7340/03M from the Hong Kong Research Grants Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Biochemistry, 3/F Laboratory Block, Faculty of Medicine Bldg., 21 Sassoon Rd., Hong Kong, China. Tel.: 852-2819-9491; Fax: 852-2855-1254; E-mail: dyjin{at}hkucc.hku.hk.

1 The abbreviations used are: ROS, reactive oxygen species; DCF-DA, 2',7'-dichlorofluorescein diacetate; RNS, reactive nitrogen species; DAF-DA, 4',5'-diaminofluorescein diacetate; DTNB, dithionitrobenzoic acid; SNP, sodium nitroprusside; CDNB, 1-chloro-2,4-dinitrobenzene; YPD, yeast extract/peptone/dextrose; tBHP, t-butyl-hydroperoxide; PN, peroxynitrite; AMA, antimycin A. Back


   ACKNOWLEDGMENTS
 
We thank K. T. Chin, Y. P. Ching, A. C. S. Chun, K. H. Kok, and M. L. Yeung for critical reading of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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