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. 2005 Jun 21;102(25):8875-80.
doi: 10.1073/pnas.0503251102. Epub 2005 Jun 13.

A cysteine-sulfinic acid in peroxiredoxin regulates H2O2-sensing by the antioxidant Pap1 pathway

Ana P Vivancos  1 , Esther A Castillo, Benoît Biteau, Carine Nicot, José Ayté, Michel B Toledano, Elena Hidalgo
Affiliations

A cysteine-sulfinic acid in peroxiredoxin regulates H2O2-sensing by the antioxidant Pap1 pathway

Ana P Vivancos et al. Proc Natl Acad Sci U S A. .

Abstract

The Schizosaccharomyces pombe transcription factor Pap1 regulates antioxidant-gene transcription in response to H2O2. Pap1 activation occurs only at low, but not elevated, H2O2 concentrations that instead strongly trigger the mitogen-activated protein kinase Sty1 pathway. Here, we identify the peroxiredoxin Tpx1 as the upstream activator of Pap1. We show that, at low H2O2 concentrations, this oxidant scavenger can transfer a redox signal to Pap1, whereas higher concentrations of the oxidant inhibit the Tpx1-Pap1 redox relay through the temporal inactivation of Tpx1 by oxidation of its catalytic cysteine to a sulfinic acid. This cysteine modification can be reversed by the sulfiredoxin Srx1, its expression in response to high doses of H2O2 strictly depending on active Sty1. Thus, Tpx1 oxidation to the cysteine-sulfinic acid and its reversion by Srx1 constitutes a previously uncharacterized redox switch in H2O2 signaling, restricting Pap1 activation within a narrow range of H2O2 concentrations.

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Figures

Fig. 1.
Fig. 1.
The peroxiredoxin Tpx1 is required for Pap1 oxidation activation by H2O2.(A) Survival of different strains in response to H2O2 exposure. Strains HM123 (WT), AM004 (Δpap1), EA38 (Δsrx1), EA49 (Δpmp20), EA37 (Δgpx1), and wild-type PN513, transformed with plasmid p120.42x (psrx1.42x), were grown in minimal media (EMM) to a final OD600 of 0.5, and the number of cells indicated at the top of the panels were spotted onto EMM plates containing or not containing H2O2. Plates were incubated at 30°C for 3–4 days. (B) Oxidation of Pap1 at different concentrations of H2O2 in different strain backgrounds. The redox state of Pap1 (with TCA extracts resolved in nonreducing SDS/PAGE followed by Western blot analysis) was determined in cells treated with H2O2 for the concentrations and times indicated. The strains used in this experiment were HM123 (WT), NT224 (sty1-1) and EA37 (Δgpx1). (C) A Δtpx1 strain transformed with a plasmid containing the nmt-driven HA-tpx1 gene (strain EA40hap-p123.41x) was incubated with thiamine for 5 h (thiamine-nonrepressive conditions; with HA-Tpx1) or 25 h (thiamine-repressive conditions; without HA-Txp1). Cells were left untreated or were treated with 0.2 or 1 mM H2O2 for the times indicated. Total HA-Tpx1 was detected in TCA extracts by reducing SDS/PAGE and Western blot analysis. Reduced and oxidized Pap1 were detected in TCA extracts by nonreducing SDS/PAGE, followed by Western blot analysis.
Fig. 2.
Fig. 2.
Pap1 and Tpx1 interact in vivo. (A) HA-Tpx1 interacts with MBP-Pap1. Five minutes after 0.2 mM H2O2 treatment, cell lysates were prepared from a Δpap1 strain (EHH108) transformed with plasmid p122.41x (pMBP-pap1) or the control vector pRep.41x, and plasmid p123.41x (pHA-tpx1). MBP-Pap1, purified from cell extracts by using amylose resin, was analyzed by reducing SDS/PAGE and Western blot with anti-Pap1 or anti-HA antibodies. As a control, total extracts of both strains were also analyzed. (B) A Δtpx1 strain transformed with a plasmid containing the nmt-driven HA-tpx1 gene (strain AV36hap-p123.42x) was transformed with a linearized plasmid containing the tpx1 promoter fused to wild-type (p145) or mutated (p145.C48S or p145.C169S) tpx1 alleles. The resulting strains, with the tpx1 alleles integrated at the leu1 locus, were incubated with thiamine for 25 h to block expression of plasmid-encoded but not chromosomally encoded versions of Tpx1 to yield cells expressing wild-type Tpx1 (Tpx1 WT) or the mutant Tpx1.C48S or Tpx1.C169S forms, as indicated. Cell treatments (0.2 mM H2O2), reducing (with DTT) or not reducing (without DTT) SDS/PAGE, and Western blot analysis with anti-Pap1 antibodies were performed as described in Fig. 1B. The positions and mass (kDa) of markers are indicated. (C) Oxidation of wild-type and mutant forms of Pap1. Strains EHH14 (expressing wild-type GFP-Pap1, lanes 1 and 2), EHH14.C278A (expressing GFP-Pap1.C278A, lanes 3 and 4), EHH14.C501A (expressing GFP-Pap1.C501A, lanes 5 and 6), EHH14.C523A (expressing GFP-Pap1.C523A, lanes 7 and 8), EHH14. C532T (expressing GFP-Pap1. C532T, lanes 9 and 10), EHH14.C501,532A (expressing GFP-Pap1.C501,532A, lanes 11 and 12), and EHH14.C501,523A,C532T (expressing GFP-Pap1.C501,523A,C532T, lanes 13 and 14), were treated (+) or not treated (–) for 5 min with 0.2 mM H2O2, as indicated. Extracts were prepared and processed as described in Fig. 1B.(D) Δpap1 strain EHH108 transformed with p122.41x (pMBP-pap1) and p123.42x (pHA-tpx1) (lanes 1 and 2), p122.41x.C278A (pMBP-pap1.C278A) and p123.42x.C169S (pHA-tpx1.C169S) (lanes 3 and 4), or p122.41x.C501A,C523A,C532T (pMBP-pap1.C501A,C523A,C532T) and p123.42x.C169S (pHA-tpx1.C169S) (lanes 5 and 6) were treated (+) or not (–) with 0.2 mM H2O2 for 5 min. TCA cell extracts, SDS/PAGE and Western blot analysis were performed as described in Fig. 1B.
Fig. 3.
Fig. 3.
The sulfiredoxin Srx1 is required for Tpx1-mediated activation of Pap1 upon severe H2O2 stress. (A and B) Formation of cysteine sulfinic acid in Tpx1 upon severe H2O2 treatment. (A) 2D PAGE analysis followed by immunoblot analysis with anti-HA antibody of reduced (SH) and oxidized (SO2H) forms of HA-Tpx1 in wild-type (Left) and Δsrx1 (Right) cells exposed to 1 mM H2O2 for the times indicated. (B) AMS alkylation of Tpx1. Cell cultures were treated with 0.2 or 1 mM H2O2, or left untreated, for the indicated times. We performed Western blot analysis of TCA extracts after in vitro alkylation of thiols (SH) but not of cysteine sulfinic acid (SO2H) residues with AMS. (C) Tpx1 reactivation and dimer formation after severe H2O2 stress depends on Srx1. The redox state (monomer versus covalent dimer) of HA-Tpx1 was determined in TCA extracts of cells treated or not treated with H2O2 for the times indicated. We performed nonreducing SDS/PAGE and Western blot analysis by using anti-HA antibody. Strains used were wild-type HM123 (WT), EA38 (Δsrx1), and AM001 (Δsty1; only in B and C), each one of them transformed with plasmid p123.81x (pHA-tpx1) to express low levels of the fusion protein HA-Tpx1.
Fig. 4.
Fig. 4.
Srx1 expression depends on Sty1. (A) Northern blot analysis of srx1 expression. Total RNA from strains HM123 (WT), AM004 (Δpap1), AM001 (Δsty1), and JM1066 (Δatf1) was obtained from cultures treated with H2O2 for the times and concentrations indicated. (B and C) The late oxidation (B) and nuclear accumulation (C) of Pap1 after 1 mM H2O2 stress depends on Sty1 and Srx1. The redox state (with nonreducing SDS/PAGE followed by Western blot analysis) and cellular distribution (with fluorescence microscopy) of a GFP-Pap1 fusion protein was determined in cells treated with H2O2 for the concentrations and times indicated. The GFP-Pap1-expressing strains used in this experiment were EHH14 (WT), EA36 (Δsrx1), AV1 (Δsty1) and AV1 overexpressing Srx1 from plasmid p120.42x (psrx1.42x).
Fig. 5.
Fig. 5.
A model for Pap1 activation in response to low (0.2 mM) vs. high (1 mM) H2O2 stress.
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