Kazuhiro Maeta

Regulation of the Yeast Yap1p Nuclear Export Signal Is Mediated by Redox Signal-Induced Reversible Disulfide Bond Formation

ABSTRACT Yap1p, a crucial transcription factor in the oxidative stress response of Saccharomyces cerevisiae, is transported in and out of the nucleus under nonstress conditions. The nuclear export step is specifically inhibited by H2O2 or the thiol oxidant diamide, resulting in Yap1p nuclear accumulation and induction of transcription of its target genes. Here we provide evidence for sensing of H2O2 and diamide mediated by disulfide bond formation in the C-terminal cysteine-rich region (c-CRD), which contains 3 conserved cysteines and the nuclear export signal (NES). The H2O2 or diamide-induced oxidation of the c-CRD in vivo correlates with induced Yap1p nuclear localization. Both were initiated within 1 min of application of oxidative stress, before the intracellular redox status of thioredoxin and glutathione was affected. The cysteine residues in the middle region of Yap1p (n-CRD) are required for prolonged nuclear localization of Yap1p in response to H2O2 and are thus also required for maximum transcriptional activity. Using mass spectrometry analysis, the H2O2-induced oxidation of the c-CRD in vitro was detected as an intramolecular disulfide linkage between the first (Cys598) and second (Cys620) cysteine residues; this linkage could be reduced by thioredoxin. In contrast, diamide induced each pair of disulfide linkage in the c-CRD, but in this case the cysteine residues in the n-CRD appeared to be dispensable for the response. Our data provide evidence for molecular mechanisms of redox signal sensing through the thiol-disulfide redox cycle coupled with the thioredoxin system in the Yap1p NES.

Green Tea Polyphenols Function as Prooxidants To Activate Oxidative-Stress-Responsive Transcription Factors in Yeasts

ABSTRACT Epigallocatechin gallate (EGCG) is the most abundant polyphenolic flavonoid in green tea. Catechin and its derivatives, including EGCG, are widely believed to function as antioxidants. Here we demonstrate that both EGCG and green tea extract (GTE) cause oxidative stress-related responses in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe under weak alkaline conditions in terms of the activation of oxidative-stress-responsive transcription factors. GTE as well as EGCG induced the nuclear localization of Yap1 in S. cerevisiae, which was repressed by the addition of catalase but not by the addition of superoxide dismutase. The same phenomena were observed for the nucleocytoplasmic localization of Msn2 in S. cerevisiae and Pap1, a Yap1 homologue, in S. pombe. The formation of intramolecular disulfide bonds has been proposed to be crucial for the H2O2-induced nuclear localization of Yap1, and we verified the importance of cysteine residues of Yap1 in response to EGCG and GTE. Additionally, we show that EGCG and GTE produce H2O2 in a weak alkaline medium. Finally, we conclude that tea polyphenols are able to act as prooxidants to cause a response to oxidative stress in yeasts under certain conditions.

Activity of the Yap1 transcription factor in Saccharomyces cerevisiae is modulated by methylglyoxal, a metabolite derived from glycolysis.

ABSTRACT Methylglyoxal (MG) is synthesized during glycolysis, although it inhibits cell growth in all types of organisms. Hence, it has long been asked why such a toxic metabolite is synthesized in vivo. Glyoxalase I is a major enzyme detoxifying MG. Here we show that the Yap1 transcription factor, which is critical for the oxidative-stress response in Saccharomyces cerevisiae, is constitutively concentrated in the nucleus and activates the expression of its target genes in a glyoxalase I-deficient mutant. Yap1 contains six cysteine residues in two cysteine-rich domains (CRDs), i.e., three cysteine residues clustering near the N terminus (n-CRD) and the remaining three cysteine residues near the C terminus (c-CRD). We reveal that any of the three cysteine residues in the c-CRD is sufficient for MG to allow Yap1 to translocate into the nucleus and to activate the expression of its target gene. A Yap1 mutant possessing only one cysteine residue in the c-CRD but no cysteine in the n-CRD and deletion of the basic leucine zipper domain can concentrate in the nucleus with MG treatment. However, substitution of all the cysteine residues in Yap1 abolishes the ability of this transcription factor to concentrate in the nucleus following MG treatment. The redox status of Yap1 is substantially unchanged, and protein(s) interaction with Yap1 through disulfide bond is hardly detected in cells treated with MG. Collectively, neither intermolecular nor intramolecular disulfide bond formation seems to be involved in Yap1 activation by MG. Moreover, we show that nucleocytoplasmic localization of Yap1 closely correlates with growth phase and intracellular MG level. We propose a novel regulatory pathway underlying Yap1 activation by a natural metabolite in the cell.

Glyoxalase system in yeasts: Structure, function, and physiology

The glyoxalase system consists of glyoxalase I and glyoxalase II. Glyoxalase I catalyzes the conversion of methylglyoxal (CH(3)COCHO), a metabolite derived from glycolysis, with glutathione to S-D-lactoylglutathione, while glyoxalase II hydrolyses this glutathione thiolester to D-lactic acid and glutathione. Since methylglyoxal is toxic due to its high reactivity, the glyoxalase system is crucial to warrant the efficient metabolic flux of this reactive aldehyde. The budding yeast Saccharomyces cerevisiae has the sole gene (GLO1) encoding the structural gene for glyoxalase I. Meanwhile, this yeast has two isoforms of glyoxalase II encoded by GLO2 and GLO4. The expression of GLO1 is regulated by Hog1 mitogen-activated protein kinase and Msn2/Msn4 transcription factors under highly osmotic stress conditions. The physiological significance of GLO1 expression in response to osmotic stress is to combat the increase in the levels of methylglyoxal in cells during the production of glycerol as a compatible osmolyte. Deficiency in GLO1 in S. cerevisiae causes pleiotropic phenotypes in terms of stress response, because the steady state level of methylglyoxal increases in glo1Δ cells thereby constitutively activating Yap1 transcription factor. Yap1 is crucial for oxidative stress response, although methylglyoxal per se does not enhance the intracellular oxidation level in yeast, but it directly modifies cysteine residues of Yap1 that are critical for the nucleocytoplasmic localization of this b-ZIP transcription factor. Consequently, glyoxalase I can be defined as a negative regulator of Yap1 through modulating the intracellular methylglyoxal level.

Regulation of the yeast phospholipid hydroperoxide glutathione peroxidase GPX2 by oxidative stress is mediated by Yap1 and Skn7

The GPX2 gene encodes a homologue of phospholipid hydroperoxide glutathione peroxidase in Saccharomyces cerevisiae. The GPX2 promoter contains three elements the sequence of which is completely consistent with the optimal sequence for the Yap1 response element (YRE). Here, we identify the intrinsic YRE that functions in the oxidative stress response of GPX2. In addition, we discovered a cis‐acting element (5′‐GGCCGGC‐3′) within the GPX2 promoter proximal to the functional YRE that is necessary for H2O2‐induced expression of GPX2. We present evidence showing that Skn7 is necessary for the oxidative stress response of GPX2 and is able to bind to this sequence. We determine the optimal sequence for Skn7 to regulate GPX2 under conditions of oxidative stress to be 5′‐GGC(C/T)GGC‐3′, and we designate this sequence the oxidative stress‐responsive Skn7 response element.

Acetylation regulates subcellular localization of eukaryotic translation initiation factor 5A (eIF5A)

Eukaryotic translation initiation factor 5A (eIF5A) is a protein subject to hypusination, which is essential for its function. eIF5A is also acetylated, but the role of that modification is unknown. Here, we report that acetylation regulates the subcellular localization of eIF5A. We identified PCAF as the major cellular acetyltransferase of eIF5A, and HDAC6 and SIRT2 as its major deacetylases. Inhibition of the deacetylases or impaired hypusination increased acetylation of eIF5A, leading to nuclear accumulation. As eIF5A is constitutively hypusinated under physiological conditions, we suggest that reversible acetylation plays a major role in controlling the subcellular localization of eIF5A.

Distinct regulatory mechanism of yeast GPX2 encoding phospholipid hydroperoxide glutathione peroxidase by oxidative stress and a calcineurin/Crz1‐mediated Ca2+ signaling pathway

The GPX2 gene encodes a homologue of mammalian phospholipid hydroperoxide glutathione peroxidase in Saccharomyces cerevisiae. Previously, we have reported that the oxidative stress‐induced expression of GPX2 is strictly regulated by Yap1 and Skn7 transcription factors. Here, we found that the expression of GPX2 is induced by CaCl2 in a calcineurin (CN)/Crz1‐dependent manner, and the CN‐dependent response element was specified in the GPX2 promoter. Neither Yap1 nor Skn7 was required for Ca2+‐dependent induction of GPX2, therefore, distinct regulation for the oxidative stress response and Ca2+ signal response for GPX2 exists in yeast cells.

Calcineurin/Crz1 destabilizes Msn2 and Msn4 in the nucleus in response to Ca(2+) in Saccharomyces cerevisiae.

Although methylglyoxal is derived from glycolysis, it has adverse effects on cellular function. Hence, the intrinsic role of methylglyoxal in vivo remains to be determined. Glyoxalase 1 is a pivotal enzyme in the metabolism of methylglyoxal in all types of organisms. To learn about the physiological roles of methylglyoxal, we have screened conditions that alter the expression of the gene encoding glyoxalase 1, GLO1, in Saccharomyces cerevisiae. We show that the expression of GLO1 is induced following treatment with Ca2+ and is dependent on the MAPK (mitogen-activated protein kinase) Hog1 protein and the Msn2/Msn4 transcription factors. Intriguingly, the Ca2+-induced expression of GLO1 was enhanced in the presence of FK506, a potent inhibitor of calcineurin. Consequently, the Ca2+-induced expression of GLO1 in a mutant that is defective in calcineurin or Crz1, the sole transcription factor downstream of calcineurin, was much greater than that in the wild-type strain even without FK506. This phenomenon was dependent upon a cis-element, the STRE (stress-response element), in the promoter that is able to mediate the response to Ca2+ signalling together with Hog1 and Msn2/Msn4. The level of Ca2+-induced expression of GLO1 reached a maximum in cells overexpressing MSN2 even when FK506 was not present, whereas in cells overexpressing CRZ1 the level was greatly reduced and increased markedly when FK506 was present. We also found that the levels of Msn2 and Msn4 proteins in Ca2+-treated cells decreased gradually and that FK506 blocked the degradation of Msn2/Msn4. We propose that Crz1 destabilizes Msn2/Msn4 in the nuclei of cells in response to Ca2+ signalling.

Role of Gcn4 for adaptation to methylglyoxal in Saccharomyces cerevisiae: Methylglyoxal attenuates protein synthesis through phosphorylation of eIF2α

Methylglyoxal is a ubiquitous 2-oxoaldehyde derived from glycolysis. Although an endogenous metabolite, methylglyoxal at high concentrations has deleterious effects on cellular functions. Since pretreatment of Saccharomyces cerevisiae cells with methylglyoxal at a low concentration alleviates the toxicity of a subsequent lethal concentration of this 2-oxoaldehyde, proteins synthesized during treatment with methylglyoxal are necessary for adaptation to methylglyoxal. Nevertheless, here we show that methylglyoxal attenuates the rate of overall protein synthesis in S. cerevisiae. Phosphorylation of the alpha subunit of translation initiation factor 2 (eIF2alpha) is induced by several types of environmental stress, and subsequently, overall protein synthesis is reduced due to the impairment of the formation of a translation initiation complex. We found that methylglyoxal activates the protein kinase Gcn2 to phosphorylate eIF2alpha. The transcription factor Gcn4 is a master regulator of gene expression under conditions of amino acid starvation and some environmental stresses, the level of which is regulated by Gcn2. We found that adaptation to methylglyoxal was impaired in gcn4Delta cells, indicating the expression of certain genes regulated by Gcn4 to be important for the adaptive response to methylglyoxal.

The Role of Acetylation in the Subcellular Localization of an Oncogenic Isoform of Translation Factor eIF5A

Mammalian cells express two isoforms of eIF5A, eIF5A1 and eIF5A2, but little is known about the function of eIF5A2. Here we report that eIF5A2 is reversibly acetylated at lysine-47. HDAC6 and SIRT2 were identified as the enzymes responsible for deacetylating eIF5A2. Analysis using acetylation-deficient mutants indicated that acetylation regulates the subcellular localization of eIF5A2.