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The transcription factors Pap1 and Prr1 collaborate to activate antioxidant, but not drug tolerance, genes in response to H2O2.

Calvo IA, García P, Ayté J, Hidalgo E - Nucleic Acids Res. (2012)

Bottom Line: Oxidation and nuclear accumulation of Pap1 can also be accomplished by genetic inhibition of thioredoxin reductase.Furthermore, genetic alteration of the nuclear export pathway, or mutations in Pap1 nuclear export signal trigger nuclear accumulation of reduced Pap1.We show here that a subset of Pap1-dependent genes, such as those coding for the efflux pump Caf5, the ubiquitin-like protein Obr1 or the dehydrogenase SPCC663.08c, only require nuclear Pap1 for activation, whereas another subset of genes, those coding for the antioxidants catalase, sulfiredoxin or thioredoxin reductase, do need oxidized Pap1 to form a heterodimer with the constitutively nuclear transcription factor Prr1.

View Article: PubMed Central - PubMed

Affiliation: Oxidative Stress and Cell Cycle Group, Department de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, C/Dr. Aiguader 88, E-08003 Barcelona, Spain.

ABSTRACT
In response to hydrogen peroxide (H(2)O(2)), the transcription factor Pap1 from Schizosaccharomyces pombe regulates transcription of genes required for adaptation to oxidative stress and for tolerance to toxic drugs. H(2)O(2) induces oxidation of Pap1, its nuclear accumulation and expression of more than fifty Pap1-dependent genes. Oxidation and nuclear accumulation of Pap1 can also be accomplished by genetic inhibition of thioredoxin reductase. Furthermore, genetic alteration of the nuclear export pathway, or mutations in Pap1 nuclear export signal trigger nuclear accumulation of reduced Pap1. We show here that a subset of Pap1-dependent genes, such as those coding for the efflux pump Caf5, the ubiquitin-like protein Obr1 or the dehydrogenase SPCC663.08c, only require nuclear Pap1 for activation, whereas another subset of genes, those coding for the antioxidants catalase, sulfiredoxin or thioredoxin reductase, do need oxidized Pap1 to form a heterodimer with the constitutively nuclear transcription factor Prr1. The ability of Pap1 to bind and activate drug tolerance promoters is independent on Prr1, whereas its affinity for the antioxidant promoters is significantly enhanced upon association with Prr1. This finding suggests that the activation of both antioxidant and drug resistance genes in response to oxidative stress share a common inducer, H(2)O(2), but alternative effectors.

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Genetic inhibition of Pap1 export renders a transcription factor insensitive to H2O2 stress. (A) Schematic representation of Pap1 activation by H2O2. Upon peroxide stress, Tpx1 mediates disulfide bond formation in Pap1, which hinders the recognition by the exportin Crm1 and its cofactor Hba1 to the Pap1 NES. Nuclear accumulation of oxidized Pap1 triggers transcription both antioxidant and drug resistance genes. The relative position of the seven cysteines residues (C) in Pap1 is indicated. (B) Localization of Pap1 in wild-type and mutant strains. The cellular distribution of GFP-Pap1 was determined by fluorescence microscopy in EHH14 (WT), EHH14.C523D (Pap1.C523D), AV19 (Δtrr1) and EA33 (Δhba1) treated or not with 0.2 mM H2O2 for 5 min. (C) Constitutively nuclear Pap1 confers resistant to caffeine but not to H2O2. Strains IC2 (WT), IC1 (Δpap1), NG25 (Δtrr1), IC2.C523D (pap1.C523D) and caf1::ura4+ (Δhba1) were grown in liquid YE media, and the indicated number of cells were spotted onto plates with or without 2 mM H2O2 or 15 mM caffeine. (D) In vivo oxidation of Pap1 in wild-type and mutant strains. Strains IC2 (WT), NG25 (Δtrr1), IC2.C523D (pap1.C523D) and caf1::ura4+ (Δhba1) were treated or not with 0.2 mM H2O2 for 5 min. TCA extracts were processed by non-reducing SDS–PAGE and analysed by western blot with antibodies against Pap1. Reduced/inactive (red. Pap1) and oxidized/active (ox. Pap1) Pap1 forms are indicated with arrows.
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gks141-F1: Genetic inhibition of Pap1 export renders a transcription factor insensitive to H2O2 stress. (A) Schematic representation of Pap1 activation by H2O2. Upon peroxide stress, Tpx1 mediates disulfide bond formation in Pap1, which hinders the recognition by the exportin Crm1 and its cofactor Hba1 to the Pap1 NES. Nuclear accumulation of oxidized Pap1 triggers transcription both antioxidant and drug resistance genes. The relative position of the seven cysteines residues (C) in Pap1 is indicated. (B) Localization of Pap1 in wild-type and mutant strains. The cellular distribution of GFP-Pap1 was determined by fluorescence microscopy in EHH14 (WT), EHH14.C523D (Pap1.C523D), AV19 (Δtrr1) and EA33 (Δhba1) treated or not with 0.2 mM H2O2 for 5 min. (C) Constitutively nuclear Pap1 confers resistant to caffeine but not to H2O2. Strains IC2 (WT), IC1 (Δpap1), NG25 (Δtrr1), IC2.C523D (pap1.C523D) and caf1::ura4+ (Δhba1) were grown in liquid YE media, and the indicated number of cells were spotted onto plates with or without 2 mM H2O2 or 15 mM caffeine. (D) In vivo oxidation of Pap1 in wild-type and mutant strains. Strains IC2 (WT), NG25 (Δtrr1), IC2.C523D (pap1.C523D) and caf1::ura4+ (Δhba1) were treated or not with 0.2 mM H2O2 for 5 min. TCA extracts were processed by non-reducing SDS–PAGE and analysed by western blot with antibodies against Pap1. Reduced/inactive (red. Pap1) and oxidized/active (ox. Pap1) Pap1 forms are indicated with arrows.

Mentions: In general, three different strategies have been described to confer Pap1-dependent resistance to several drugs (Figure 1A): (i) constitutive oxidation of Pap1 by deletion of the thioredoxin reductase gene; in this particular case, Pap1 export is blocked since the protein is locked in the oxidized conformation; (ii) over-expression of the protein, with concomitant enhancement of Pap1-dependent transcription; this can be genetically accomplished by depletion of a subunit of the 26S proteasome, Pad1 (16,18), or deletion of the ubr1 gene, encoding a ubiquitin ligase which regulates nuclear Pap1 stability (19); (iii) inhibition of Pap1 nuclear export, either by chemical- (9) or temperature-(10) dependent inactivation of the essential Crm1 protein, or by depletion of the Crm1 cofactor Hba1 (20). We demonstrate here that, unexpectedly, this gain of drug resistance does not correlate with enhanced tolerance to oxidative stress. In fact, defects in nuclear export render Pap1 being insensitive to H2O2-mediated oxidation, probably due to the cytoplasmic localization of its upstream redox transmitter, Tpx1. Analysis of the transcriptome of these constitutively nuclear-expressing cell types indicate that the drug tolerance genes are being activated under basal conditions, but not the antioxidant ones. In fact, we have determined that only oxidized nuclear Pap1, but not the reduced one, interacts with the transcription regulator Prr1 and activates also antioxidant genes. The distinct regulation of these two subsets of genes may reflect an evolutionary merge of previous and independent oxidative stress and multidrug resistance responses.Figure 1.


The transcription factors Pap1 and Prr1 collaborate to activate antioxidant, but not drug tolerance, genes in response to H2O2.

Calvo IA, García P, Ayté J, Hidalgo E - Nucleic Acids Res. (2012)

Genetic inhibition of Pap1 export renders a transcription factor insensitive to H2O2 stress. (A) Schematic representation of Pap1 activation by H2O2. Upon peroxide stress, Tpx1 mediates disulfide bond formation in Pap1, which hinders the recognition by the exportin Crm1 and its cofactor Hba1 to the Pap1 NES. Nuclear accumulation of oxidized Pap1 triggers transcription both antioxidant and drug resistance genes. The relative position of the seven cysteines residues (C) in Pap1 is indicated. (B) Localization of Pap1 in wild-type and mutant strains. The cellular distribution of GFP-Pap1 was determined by fluorescence microscopy in EHH14 (WT), EHH14.C523D (Pap1.C523D), AV19 (Δtrr1) and EA33 (Δhba1) treated or not with 0.2 mM H2O2 for 5 min. (C) Constitutively nuclear Pap1 confers resistant to caffeine but not to H2O2. Strains IC2 (WT), IC1 (Δpap1), NG25 (Δtrr1), IC2.C523D (pap1.C523D) and caf1::ura4+ (Δhba1) were grown in liquid YE media, and the indicated number of cells were spotted onto plates with or without 2 mM H2O2 or 15 mM caffeine. (D) In vivo oxidation of Pap1 in wild-type and mutant strains. Strains IC2 (WT), NG25 (Δtrr1), IC2.C523D (pap1.C523D) and caf1::ura4+ (Δhba1) were treated or not with 0.2 mM H2O2 for 5 min. TCA extracts were processed by non-reducing SDS–PAGE and analysed by western blot with antibodies against Pap1. Reduced/inactive (red. Pap1) and oxidized/active (ox. Pap1) Pap1 forms are indicated with arrows.
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gks141-F1: Genetic inhibition of Pap1 export renders a transcription factor insensitive to H2O2 stress. (A) Schematic representation of Pap1 activation by H2O2. Upon peroxide stress, Tpx1 mediates disulfide bond formation in Pap1, which hinders the recognition by the exportin Crm1 and its cofactor Hba1 to the Pap1 NES. Nuclear accumulation of oxidized Pap1 triggers transcription both antioxidant and drug resistance genes. The relative position of the seven cysteines residues (C) in Pap1 is indicated. (B) Localization of Pap1 in wild-type and mutant strains. The cellular distribution of GFP-Pap1 was determined by fluorescence microscopy in EHH14 (WT), EHH14.C523D (Pap1.C523D), AV19 (Δtrr1) and EA33 (Δhba1) treated or not with 0.2 mM H2O2 for 5 min. (C) Constitutively nuclear Pap1 confers resistant to caffeine but not to H2O2. Strains IC2 (WT), IC1 (Δpap1), NG25 (Δtrr1), IC2.C523D (pap1.C523D) and caf1::ura4+ (Δhba1) were grown in liquid YE media, and the indicated number of cells were spotted onto plates with or without 2 mM H2O2 or 15 mM caffeine. (D) In vivo oxidation of Pap1 in wild-type and mutant strains. Strains IC2 (WT), NG25 (Δtrr1), IC2.C523D (pap1.C523D) and caf1::ura4+ (Δhba1) were treated or not with 0.2 mM H2O2 for 5 min. TCA extracts were processed by non-reducing SDS–PAGE and analysed by western blot with antibodies against Pap1. Reduced/inactive (red. Pap1) and oxidized/active (ox. Pap1) Pap1 forms are indicated with arrows.
Mentions: In general, three different strategies have been described to confer Pap1-dependent resistance to several drugs (Figure 1A): (i) constitutive oxidation of Pap1 by deletion of the thioredoxin reductase gene; in this particular case, Pap1 export is blocked since the protein is locked in the oxidized conformation; (ii) over-expression of the protein, with concomitant enhancement of Pap1-dependent transcription; this can be genetically accomplished by depletion of a subunit of the 26S proteasome, Pad1 (16,18), or deletion of the ubr1 gene, encoding a ubiquitin ligase which regulates nuclear Pap1 stability (19); (iii) inhibition of Pap1 nuclear export, either by chemical- (9) or temperature-(10) dependent inactivation of the essential Crm1 protein, or by depletion of the Crm1 cofactor Hba1 (20). We demonstrate here that, unexpectedly, this gain of drug resistance does not correlate with enhanced tolerance to oxidative stress. In fact, defects in nuclear export render Pap1 being insensitive to H2O2-mediated oxidation, probably due to the cytoplasmic localization of its upstream redox transmitter, Tpx1. Analysis of the transcriptome of these constitutively nuclear-expressing cell types indicate that the drug tolerance genes are being activated under basal conditions, but not the antioxidant ones. In fact, we have determined that only oxidized nuclear Pap1, but not the reduced one, interacts with the transcription regulator Prr1 and activates also antioxidant genes. The distinct regulation of these two subsets of genes may reflect an evolutionary merge of previous and independent oxidative stress and multidrug resistance responses.Figure 1.

Bottom Line: Oxidation and nuclear accumulation of Pap1 can also be accomplished by genetic inhibition of thioredoxin reductase.Furthermore, genetic alteration of the nuclear export pathway, or mutations in Pap1 nuclear export signal trigger nuclear accumulation of reduced Pap1.We show here that a subset of Pap1-dependent genes, such as those coding for the efflux pump Caf5, the ubiquitin-like protein Obr1 or the dehydrogenase SPCC663.08c, only require nuclear Pap1 for activation, whereas another subset of genes, those coding for the antioxidants catalase, sulfiredoxin or thioredoxin reductase, do need oxidized Pap1 to form a heterodimer with the constitutively nuclear transcription factor Prr1.

View Article: PubMed Central - PubMed

Affiliation: Oxidative Stress and Cell Cycle Group, Department de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, C/Dr. Aiguader 88, E-08003 Barcelona, Spain.

ABSTRACT
In response to hydrogen peroxide (H(2)O(2)), the transcription factor Pap1 from Schizosaccharomyces pombe regulates transcription of genes required for adaptation to oxidative stress and for tolerance to toxic drugs. H(2)O(2) induces oxidation of Pap1, its nuclear accumulation and expression of more than fifty Pap1-dependent genes. Oxidation and nuclear accumulation of Pap1 can also be accomplished by genetic inhibition of thioredoxin reductase. Furthermore, genetic alteration of the nuclear export pathway, or mutations in Pap1 nuclear export signal trigger nuclear accumulation of reduced Pap1. We show here that a subset of Pap1-dependent genes, such as those coding for the efflux pump Caf5, the ubiquitin-like protein Obr1 or the dehydrogenase SPCC663.08c, only require nuclear Pap1 for activation, whereas another subset of genes, those coding for the antioxidants catalase, sulfiredoxin or thioredoxin reductase, do need oxidized Pap1 to form a heterodimer with the constitutively nuclear transcription factor Prr1. The ability of Pap1 to bind and activate drug tolerance promoters is independent on Prr1, whereas its affinity for the antioxidant promoters is significantly enhanced upon association with Prr1. This finding suggests that the activation of both antioxidant and drug resistance genes in response to oxidative stress share a common inducer, H(2)O(2), but alternative effectors.

Show MeSH
Related in: MedlinePlus