Volume 45, Issue 3, Pages 398-408 (February 2012) Inactivation of a Peroxiredoxin by Hydrogen Peroxide Is Critical for Thioredoxin- Mediated Repair of Oxidized Proteins and Cell Survival  Alison M. Day, Jonathon D. Brown, Sarah R. Taylor, Jonathan D. Rand, Brian A. Morgan, Elizabeth A. Veal  Molecular Cell  Volume 45, Issue 3, Pages 398-408 (February 2012) DOI: 10.1016/j.molcel.2011.11.027 Copyright © 2012 Elsevier Inc. Terms and Conditions

Molecular Cell 2012 45, 398-408DOI: (10.1016/j.molcel.2011.11.027) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 The Thioredoxin Peroxidase Activity of Eukaryotic Typical 2-Cys Prx, Such as Tpx1, Is Sensitive to Hydrogen Peroxide-Induced Inactivation by Hyperoxidation of the Peroxidatic Cysteine In the catalytic breakdown of hydrogen peroxide by Prx, such as Tpx1, the sulfenic (SOH) derivative of the peroxidatic cysteine (Cys47) is stabilized by the formation of a disulfide bond with the resolving cysteine (Cys169) on a neighboring molecule (Tpx1-S-S-Tpx1). This disulfide is reduced by the oxidoreductase thioredoxin (Trx1) via the formation of a mixed disulfide intermediate (Trx1-S-S-Tpx1), which is resolved by formation of an intramolecular disulfide in Trx1 and reduction of Tpx1. Thioredoxin reductase uses NADPH as an electron donor to reduce Trx1. However, if hydrogen peroxide reacts with the sulfenic derivative of Prx (Tpx1-SOH), then it will become hyperoxidized to sulfinic (Tpx1-SOOH). The free peroxidatic cysteine in the Tpx1 single disulfide form is particularly sensitive to hyperoxidation (Figure S3) (Jara et al., 2007). In yeast, plants and humans sulfiredoxin, Srx1, catalyzes the reduction of hyperoxidized Prx, restoring thioredoxin peroxidase activity. Molecular Cell 2012 45, 398-408DOI: (10.1016/j.molcel.2011.11.027) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 The Thioredoxin Trx1 Is Important for Reduction of Tpx1 and Pap1 (A) Immunostaining of mid-log phase growing S. pombe cells (JB35) with anti-Tpx1 or anti-FLAG antibodies revealed that Tpx1 and FLAG-Trx1 are predominantly cytoplasmic and excluded from the nucleus (DAPI stained). (B) Analysis of the redox status of AMS-treated Tpx1, from wild-type (SB3) and Δtrx1 (EV79) cells treated for 10 min with 0, 0.2, 1, and 6 mM hydrogen peroxide, revealed that Trx1 is important for reduction of Tpx1-Tpx1 disulfides (α-Tpx1) and hyperoxidation of Tpx1 (α-Prx-SO3). (C) Analysis of the redox status of Pk epitope-tagged Pap1 (Pap1-Pk), from wild-type (SB3) and Δtrx1 (EV79) cells treated for 10 min with 0, 0.2, 1, and 6 mM hydrogen peroxide, revealed that, in the absence of Trx1, Pap1 is constitutively partially oxidized to a similar extent as Pap1 in wild-type cells treated with 0.2 mM hydrogen peroxide. (D) Visualization of Pap1-Pk by indirect immunofluorescence using anti-Pk antibody in wild-type (SB3) and Δtrx1 (EV79) cells treated for 10 min with the indicated concentrations of hydrogen peroxide revealed that Pap1-Pk is constitutively nuclear in the absence of Trx1 (see also Figure S1). Molecular Cell 2012 45, 398-408DOI: (10.1016/j.molcel.2011.11.027) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 In Hydrogen Peroxide-Treated Cells the Thioredoxin Peroxidase Tpx1 Is the Major Substrate for the Oxidoreductase Activity of Trx1 The redox status of FLAG-Trx1 in (A and B) wild-type (WT; CHP429) or (A) Δtpx1 (VX00) cells containing Rep1FLAGTrx1, or (C) Δtpx1 cells (EV45) containing Rep2FLAGTrx1 and either Rep1 vector, Rep1tpx1+, or Rep1tpx1C169S, before and after treatment with 0.2, 1, or 6 mM hydrogen peroxide was assessed by SDS PAGE and western blotting (α-FLAG) of proteins treated, as indicated, with AMS. Oxidized and reduced monomeric FLAG-Trx1 were separated, based on their relative mobilities after reaction of any reduced cysteine thiols with AMS. Other oxidized forms of FLAG-Trx1 were also detected (FLAG-Trx1-containing disulfides). ∗ indicates a minor oxidized form of FLAG-Trx1 that may be glutathionylated. The nonspecific band (A) may be oxidized by hydrogen peroxide, but is also detected in cells which do not express FLAG-Trx1 (data not shown). Molecular Cell 2012 45, 398-408DOI: (10.1016/j.molcel.2011.11.027) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 Trx1 Is Required for Reduction of Mxr1 Analysis of the oxidation state of Pk epitope-tagged Mxr1 (Mxr1-Pk) expressed from its normal chromosomal locus in (A and B) wild-type (AD86) and (A) Δtrx1 (AD114) cells treated with 0.2 or 1 mM hydrogen peroxide as indicated. Western blotting with anti-Pk antibodies of proteins reacted with AMS as indicated revealed that Mxr1-Pk is oxidized to AMS-resistant forms with increased mobility (A) in Δtrx1 cells and (A and B) after treatment of wild-type cells with 0.2 mM and, to a lesser extent, 1 mM hydrogen peroxide. The detection of multiple bands in non-AMS-treated samples may reflect post-lysis thiol exchange/oxidation reactions occurring at cysteines in Mxr1 in the absence of AMS (see also Figure S2). Molecular Cell 2012 45, 398-408DOI: (10.1016/j.molcel.2011.11.027) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 Hyperoxidation of Tpx1 Allows Trx1 to Reduce Mxr1 at High Concentrations of Hydrogen Peroxide The oxidation state of AMS-treated Mxr1-Pk, Tpx1, or FLAG-Trx1 expressed from their normal chromosomal loci was examined. (A) Analysis of the oxidation state of Tpx1 (upper panel; α-PRDX-2) or FLAG-Trx1 (lower panel; α-FLAG) in cells expressing normal (WT; AD115) or high (HighTpx1WT; AD99) levels of wild-type Tpx1, or Tpx11-181 (AD97), before and after treatment with 6 mM hydrogen peroxide as indicated. (B) Analysis of the oxidation state of Mxr1-Pk in cells expressing normal (WT; AD86) or high (HighTpx1WT; AD88) levels of wild-type Tpx1, or Tpx11-181 (AD89), before and after treatment with 6 mM hydrogen peroxide as indicated. (C) Quantitative densitometric analyses of the oxidation state of Tpx1, FLAG-Trx1, and Mxr1-Pk on western blots obtained from three independent experiments similar to those described in (A) and (B) with cells treated for 0, 1, or 10 min with 6 mM hydrogen peroxide. The percentage of nonhyperoxidized Tpx1 was determined by dividing total reduced Tpx1 and Tpx1-containing disulfide signals by total Tpx1. The percentage of oxidized Trx1 was determined by dividing total FLAG-Trx1ox and FLAG-Trx1-containing disulfide signals by total FLAG-Trx1. The percentage of oxidized Mxr1 was determined by dividing the Mxr1-Pkox signal by the total Mxr1-Pk signal. Mean values are shown and, where there was variation from this mean, error bars representing the SEM are shown (see also Figure S3). Molecular Cell 2012 45, 398-408DOI: (10.1016/j.molcel.2011.11.027) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 Maintaining the Thioredoxin Peroxidase Activity of Tpx1 following Exposure to High Levels of Hydrogen Peroxide Increases Oxidative Protein Damage and Reduces Cell Survival (A and B) Proteins were analyzed by nonreducing SDS PAGE and western blotting with antibodies specific for protein-methionine sulfoxide (α−MetO) from (A) cells expressing normal (WT; AD82) and high (HighTpx1WT; JR68) levels of wild-type Tpx1 (Figures 5 and S3C), or hydrogen peroxide-resistant Tpx1 (Tpx11-181; JR20) (Figures 5, S3B, and S3C), and (B) Δtrx1 (JB30) or Δsrx1 cells, expressing high levels of wild-type Tpx1 (AD116) or Tpx11-181 (AD117), treated with 6mM hydrogen peroxide as indicated. (∗) indicates bands which increased after hydrogen peroxide treatment. In (A), several protein-methionine sulfoxide bands (∗∗) increased in intensity and duration in cells containing higher than normal levels of wild-type Tpx1 or Tpx11-181. # indicates bands which do not change after hydrogen peroxide treatment and therefore indicate loading. A prominent band (v) was generally detected but varied in intensity between lanes and experiments, with variations not reflecting differences in peroxide treatment, loading, or particular strains/samples (Figures 6A and 6B and data not shown). (C) Northern blot analysis of RNA isolated from wild-type cells (AD38) indicates that srx1+ mRNA is increased by treatment with 1 and 6 mM but not 25 mM hydrogen peroxide. leu1+ mRNA levels are shown as a loading control. (D and E) The oxidation state of (D) Tpx1 (α-Tpx1) and (E) FLAG-Trx1 (α-FLAG) in cells expressing wild-type Tpx1 and FLAG-Trx1 from their normal chromosomal loci (JB35) and containing Rep1 (vector) or Rep1-Srx1 (+Srx1) was examined before and after treatment with 25 mM hydrogen peroxide as indicated. Samples were treated with AMS as indicated. ∗ indicates a minor oxidized form of FLAG-Trx1 that may be glutathionylated. (F) The survival of wild-type cells (JB35) containing Rep1 (vector) or Rep1-Srx1 (+Srx1) was examined after treatment with 25 mM hydrogen peroxide. Error bars represent the SEM (see also Figure S4). Molecular Cell 2012 45, 398-408DOI: (10.1016/j.molcel.2011.11.027) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 7 Appropriate Responses to Hydrogen Peroxide Are Initiated by Changes in the Redox Status of Tpx1 which Determine the Activity of Trx1 toward Other Substrates Under homeostatic conditions reduced Trx1 (Trx1red) maintains substrate proteins, which include Tpx1, Mxr1, and Pap1, in a reduced state. When cells are exposed to low levels of hydrogen peroxide, Trx1 becomes more oxidized due to its involvement in the catalytic removal of hydrogen peroxide by Tpx1. Thus, Trx1-mediated reduction of other substrates is inhibited. This allows accumulation of oxidized Pap1 and hence increased antioxidant gene expression. Together, these adaptive responses (gene expression, protein repair, and Tpx1 activity) act to restore homeostasis. At high levels of hydrogen peroxide, there is increased oxidative protein damage and gene expression is inhibited. In these acute oxidative stress conditions, hyperoxidation of Tpx1 to Trx1-resistant sulfinic (SOOH) derivatives is essential to target reduced Trx1 toward other substrates, such as Mxr1, allowing the repair of oxidized proteins vital for cell survival under these conditions (Figure S2). Red arrows indicate reactions in which hydrogen peroxide participates. The thickness of each arrow gives a qualitative indication of the relative reaction rates under each condition, with dashed arrows indicating likely minimal reactions. The predominant redox forms of Pap1 and Tpx1 under each condition are indicated by bold outline and bold type face. Molecular Cell 2012 45, 398-408DOI: (10.1016/j.molcel.2011.11.027) Copyright © 2012 Elsevier Inc. Terms and Conditions