Yap1 activation by H2O2 or thiol-reactive chemicals elicits distinct adaptive gene responses

doi:10.1016/j.freeradbiomed.2010.10.697

Free Radical Biology and Medicine

Volume 50, Issue 1, 1 January 2011, Pages 1-13

https://doi.org/10.1016/j.freeradbiomed.2010.10.697 Get rights and content

Abstract

The yeast Saccharomyces cerevisiae transcription factor Yap1 mediates an adaptive response to oxidative stress by regulating protective genes. H₂O₂ activates Yap1 through the Gpx3-mediated formation of a Yap1 Cys303–Cys598 intramolecular disulfide bond. Thiol-reactive electrophiles can activate Yap1 directly by adduction to cysteine residues in the C-terminal domain containing Cys598, Cys620, and Cys629. H₂O₂ and N-ethylmaleimide (NEM) showed no cross-protection against each other, whereas another thiol-reactive chemical, acrolein, elicited Yap1-dependent cross-protection against NEM, but not H₂O₂. Either Cys620 or Cys629 was sufficient for activation of Yap1 by NEM or acrolein; Cys598 was dispensable for this activation mechanism. To determine whether Yap1 activated by H₂O₂ or thiol-reactive chemicals elicits distinct adaptive gene responses, microarray analysis was performed on the wild-type strain or its isogenic single-deletion strain Δyap1 treated with control buffer, H₂O₂, NEM, or acrolein. Sixty-five unique H₂O₂ and 327 NEM and acrolein Yap1-dependent responsive genes were identified. Functional analysis using single-gene-deletion yeast strains demonstrated that protection was conferred by CTA1 and CTT1 in the H₂O₂-responsive subset and YDR042C in the NEM- and acrolein-responsive subset. These findings demonstrate that the distinct mechanisms of Yap1 activation by H₂O₂ or thiol-reactive chemicals result in selective expression of protective genes.

Section snippets

Bacterial and yeast strains

Escherichia coli strain DH5α was used to construct, propagate, and maintain all plasmids in this study. For E. coli cells transformed with plasmids, the antibiotic ampicillin (100 μg/ml) was added to Luria broth (LB) medium (1% w/v tryptone, 0.5% w/v yeast extract, and 0.5% w/v NaCl). The yeast Saccharomyces cerevisiae strain BY4741 (MATa his3-Δ1 leu2-Δ0 met15-Δ0 ura3-Δ0) [24] and the isogenic single-gene-deletion strains (created by the Saccharomyces Genome Deletion Project: //sequence-www.stanford.edu/group/yeast_deletion_project/deletions3.html

Two Yap1-activation mechanisms lead to distinct adaptive responses

To confirm that Yap1 is responsible for the induced resistance to H₂O₂, the H₂O₂ adaptive response was measured in the wild-type yeast strain BY4741 and its isogenic YAP1 deletion strain (Δyap1). Patch assays as well as quantitative survival analyses were performed. The wild-type strain pretreated with 0.3 mM H₂O₂ for 1 h acquired higher resistance to challenge with increasing concentrations of H₂O₂, showing fairly strong growth after treatment with 10 mM H₂O₂. In contrast, nonpretreated cells

Discussion

Although both H₂O₂- and thiol-reactive chemical-induced adaptive responses are mediated by the Yap1 transcription factor, pretreatment with H₂O₂ or cysteine thiol-reactive chemicals failed to cross-protect against a subsequent challenge with the other class of inducer. To explain this selectivity, we have proposed a model of Yap1 transcriptional activation in which the specific cysteine reactivity of Yap1 leads to distinct active conformations of the transcription factor that recognize and

Acknowledgments

This work was funded by the Feinstone Chair of Molecular Biology and the W. Harry Feinstone Center for Genomic Research and supported by the facilities of the Integrated Microscopy Center (University of Memphis) and the Molecular Research Center (University of Tennessee Health Science Center).

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In contrast, Yap1 oxidation by electrophilic compounds results in the induction of genes related to xenobiotic detoxification, such as genes encoding major facilitator and ATP-binding cassette transporters (Alarco et al., 1997; Wemmie et al., 1994). Consistent with this, H2O2 and N-ethylmaleimide do not induce cross-protection against each other (Ouyang et al., 2011). The mechanism by which different oxidized forms of Yap1 regulate different sets of genes is not well understood.
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Original ContributionYap1 activation by H2O2 or thiol-reactive chemicals elicits distinct adaptive gene responses

Abstract

Section snippets

Bacterial and yeast strains

Two Yap1-activation mechanisms lead to distinct adaptive responses

Discussion

Acknowledgments

Curr. Opin. Struct. Biol.

FEBS Lett.

Cell

J. Biol. Chem.

Cell

J. Biol. Chem.

J. Biol. Chem.

Free Radic. Biol. Med.

Gene

J. Biol. Chem.

Methods

Meth. Enzymol.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

Curr. Opin. Genet. Dev.

Trends Cardiovasc. Med.

J. Biol. Chem.

Cell

J. Biol. Chem.

J. Mol. Biol.

Oxidative stress: the paradox of aerobic life

Biochem. Soc. Symp.

Inducible repair of oxidative DNA damage in Escherichia coli

Nature

Inducibility of the response of yeast cells to peroxide stress

J. Gen. Microbiol.

Redox-dependent transcriptional regulation

Circ. Res.

Saccharomyces cerevisiae exhibits a yAP-1-mediated adaptive response to malondialdehyde

J. Bacteriol.

Yap, a novel family of eight bZIP proteins in Saccharomyces cerevisiae with distinct biological functions

Mol. Cell. Biol.

Yeast YAP1 encodes a novel form of the jun family of transcriptional activator proteins

Genes Dev.

Original Contribution
Yap1 activation by H₂O₂ or thiol-reactive chemicals elicits distinct adaptive gene responses