Piecing Together How Peroxiredoxins Maintain Genomic Stability

Abstract

Peroxiredoxins, a highly conserved family of thiol oxidoreductases, play a key role in oxidant detoxification by partnering with the thioredoxin system to protect against oxidative stress. In addition to their peroxidase activity, certain types of peroxiredoxins possess other biochemical activities, including assistance in preventing protein aggregation upon exposure to high levels of oxidants (molecular chaperone activity), and the transduction of redox signals to downstream proteins (redox switch activity). Mice lacking the peroxiredoxin Prdx1 exhibit an increased incidence of tumor formation, whereas baker's yeast (Saccharomyces cerevisiae) lacking the orthologous peroxiredoxin Tsa1 exhibit a mutator phenotype. Collectively, these findings suggest a potential link between peroxiredoxins, control of genomic stability, and cancer etiology. Here, we examine the potential mechanisms through which Tsa1 lowers mutation rates, taking into account its diverse biochemical roles in oxidant defense, protein homeostasis, and redox signaling as well as its interplay with thioredoxin and thioredoxin substrates, including ribonucleotide reductase. More work is needed to clarify the nuanced mechanism(s) through which this highly conserved peroxidase influences genome stability, and to determine if this mechanism is similar across a range of species.

Keywords: genomic instability; mutator; oxidative stress; peroxiredoxin; redox switch; ribonucleotide reductase; sulfiredoxin; thiol peroxidase; thioredoxin.

Figure 1

Tsa1 and other typical 2-Cys peroxiredoxins detoxify peroxides through a thiol-dependent, disulfide-based mechanism. Upon binding a peroxide, Tsa1 is oxidized on its peroxidatic cysteine to form a cysteine sulfenic acid (–SOH). Subsequently, the sulfenic acid condenses with the thiol in the resolving cysteine to form a disulfide bond. Thioredoxins reduce the resulting disulfide bond to complete the catalytic cycle.

Figure 2

Reversible hyperoxidation of Tsa1 and other 2-Cys peroxiredoxins leads to the acquisition of molecular chaperone activity and may result in the oxidation of other proteins. During periods of pronounced oxidative stress, the thiol group in the peroxidatic cysteine of Tsa1 and similar peroxiredoxins can form cysteine sulfinic acid (–SO₂H). This hyperoxidation event can cause Tsa1 decamers to oligomerize into high molecular weight (HMW) complexes, which are thought to possess molecular chaperone activity as holdases. An indirect effect of Tsa1 inactivation is that other proteins that are normally less sensitive to peroxide become oxidized. Hyperoxidized Tsa1 can be restored to its functional form by the combined actions of the sulfiredoxin and thioredoxin systems.

Figure 3

Tsa1 may influence mutation rates in a direct and/or an indirect way. Given the three biochemical activities of Tsa1 and related peroxiredoxins (i.e., peroxide detoxification, molecular chaperone activity, and redox switch activity), it is possible that any of these directly contribute to suppressing mutations. However, there is evidence suggesting that Tsa1 may influence genomic stability by titrating thioredoxin away from other substrates involved in DNA synthesis or repair, most notably ribonucleotide reductase. In the case of the Tsa1-ribonucleotide reductase competition hypothesis, Tsa1-mediated thioredoxin (Trx) sequestration may be a way of regulating ribonucleotide reductase activity, thereby maintaining appropriate dNTP levels and lowering mutation rates.

Figure 4

The peroxidatic cysteine in Tsa1 is required to protect against H₂O₂ and lower mutation rates. (A) Western blot analysis of FLAG-tagged Tsa1 variants. Pgk1 levels were monitored as a loading control. (B) Stationary phase cultures of wild-type (BY4741) cells transformed with vector, tsa1Δ tsa2Δ cells transformed with vector, or tsa1Δ tsa2Δ cells expressing FLAG-tagged Tsa1 variants were diluted serially, plated on non-selective growth medium (YPD) or YPD containing 4 mM H₂O₂, and grown for 48 h at 30 °C. Results are representative of three independent experiments. (C) Mutation rates in cells expressing Tsa1 variants were determined by monitoring fluctuation of the CAN1 gene, as assessed by counting canavanine-resistant colonies for nine independent isolates of each strain in duplicate. The graph depicts the median mutation rate ± the 95% confidence limit for each strain. Detailed methods are available in Supplementary Materials.

Figure 5

An overexpression screen of yeast peroxidases reveals that only Tsa1 and Tsa2 protect against H₂O₂ and suppress mutations in tsa1Δ tsa2Δ yeast. (A) Western blot analysis of FLAG-tagged peroxidases. Pgk1 levels were monitored as a loading control. (B) Stationary phase cultures of wild-type (BY4741) cells transformed with vector, tsa1Δ tsa2Δ cells transformed with vector, or tsa1Δ tsa2Δ cells expressing FLAG-tagged peroxidases were diluted serially, plated on non-selective growth medium (YPD) or YPD containing 4 mM H₂O₂, and grown for 48 h at 30 °C. Results are representative of three independent experiments. (C) Mutation rates in cells expressing various peroxidases were determined by monitoring fluctuation of the CAN1 gene, as assessed by counting canavanine-resistant colonies for nine independent isolates of each strain in duplicate. The graph depicts the median mutation rate ± the 95% confidence limit for each strain. Detailed methods are available in Supplementary Materials.