Molecular evolution of the adaptive mechanisms against reactive oxygen stress in yeast

本研究的目的是获得酵母菌对氧化应激适应性反应进化的线索。为了实现这一目标，我们构建了几个缺乏抗氧化酶的酿酒酵母突变株，并分析了这些突变细胞对氧化压力的反应。人们认为微生物不存在以谷胱甘肽(GSH)为电子供体的过氧化物酶。微生物被认为利用细胞色素c作为过氧化物酶反应的电子供体(细胞色素c过氧化物酶)。在植物中，抗坏血酸是抗坏血酸过氧化物酶的电子供体；而在哺乳动物系统中，GSH被用作电子供体(谷胱甘肽过氧化物酶，GPX)。我们从mrakii中克隆了GPL1(GPX-like)基因，该基因含有一个783个碱基的开放阅读框。酿酒酵母中GPX的特性将为我们揭示生物体中对抗氧化应激的机制提供线索。我们还研究了过氧化氢酶在适应氧化应激反应中的重要性。酿酒酵母有两个过氧化氢酶基因(CTT1和CTA1)，我们对这两个基因都进行了干扰。在对数生长期，突变株ctt1 Delta/cta1DELTA对H_2O_2的敏感性与野生型几乎相同，但不能适应H_2O_2胁迫。以前我们曾报道过gsh1突变体对H_2O_2高度敏感，完全不能适应H_2O_2。此外，我们还克隆了OSR1基因，该基因可增强对脂质氢过氧化氢引起的氧化应激的抵抗力。综上所述，过氧化氢酶可能在紧急情况下发挥作用或作为替代备用，GSH可能在氧化应激反应中发挥更重要的作用。为此，我们克隆了编码谷胱甘肽合成酶的GSH2基因，该酶是谷胱甘肽生物合成的第二种酶。我们明确了GSH的生物合成对于上述对氧化胁迫的抗性和适应性反应是重要的，然后我们研究了GSH再循环对H_2O_2适应性反应的影响。GSH被氧化成谷胱甘肽二硫化物(GSSG)，在谷胱甘肽还原酶(GR)和葡萄糖-6-磷酸脱氢酶(G6PDH)的作用下被还原为GSH(还原型)。GR缺失突变体仍能适应H_2O_2胁迫，而G6PDH缺失突变体则不能。G6PDH提供NADPH，因此，该酶可能不仅为GR提供还原能力，还可能在氧化胁迫之外的其他胁迫反应中发挥关键作用。我们从酿酒酵母中克隆了乙醛酶I基因(GLO1)，并通过分析GLO1缺乏的表型以及GLO1在GSH1或GSH2背景下的过表达来研究其在酵母细胞中的生理功能。我们还阐明了在高渗透条件下GLO1基因的表达受HOG-MAPK途径的调控，因此我们推测GSH可能在酵母对环境胁迫的适应性反应中起关键作用。较少

The objective of this study is to obtain a clue of the evolution of adaptive response to oxidative stress in yeasts. To accomplish this objective, we construdted several mutants of Saccharomyces cerevisiae which lacked antioxidant enzymes and analyzed how such mutant cells respond to oxidative stess.It has been believed that microorganisms do not have peroxidases whose electron donor is glutathione (GSH). Microorganisms are believed to use cytochrome c as an electron donor for the peroxidase reaction (cytochrome c peroxidase). In plants, ascorbate is an electron donor for ascorbate peroxidase ; whereas in mammalian system, GSH is used as an electron donor (glutathione peroxidase, GPx). However, we discovered a GPx from the yeast, Hansenula mrakii. We cloned the GPL1(GPx-like)gene from H.mrakii, which contained an open reading frame with 78 3bp. We are also analyzing three GPx-homologue genes from S.cerevisiae. GPx activity was not detected in the disruptant of all of these genes, and e … More xpression of one of the genes was expressed by oxidative stress. Characterization of GPx in S.cerevisiae would give us a hint for the evolution of the mechanisms against oxidative stress in organisms.We also studied importance of catalase in adaptive response to oxidative stress. S.cerevisiae has two catalase genes(CTT1 and CTA1), and we disrupted both genes. The ctt1 DELTA/cta1DELTA double disruptant showed almost the same susceptibility to H_2O_2 compared with wild-type cell at log phase, however, such a mutant could not show adaptation to H_2O_2 stress. Previously, we reported that gsh1 mutant was hypersensitive to H_2O_2, and could not adapt to H_2O_2 at all. Furthermore, we have cloned the OSR1 gene which enhanced resistance against oxidative stress caused by lipid hydroperoxide. Intracellular GSH level increased in the OSR1-overexpressing cell. Taken together, catalase may be functioning at emergency or as an alternative spare, and GSH is likely to play more important role in oxidative stress response. To condirm this, we cloned the GSH2 gene encofing glutathione synthetase, the second enzyme for glutathione biosynthesis. The gsh2 DELTA mutant was also hypersensitive to oxidative stress.We cold clarity that biosynthesis of GSH was important fot resistance as well as adaptive response to oxidative stress as described above, We then investigated effect of GSH-recysling on adaptive response to H_2O_2. GSH is oxidized to glutathione disulfide (GSSG), and reduced to GSH (reduced form) by the actions of glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PDH). We disrupted cach gene and analyzed their phenotype regarding to oxidative stress response. GR-deficient mutant still could show adapttaion to H_2O_2 stress, however, the G6PDH-deficient mutant could not. G6PDH supplies NADPH,therefore, the enzyme may be playing some roles not only supplying a reducing power to GR but also other enzymes which may have acritical function in other stress responses in addition to oxidative stress.In addition to there enzymes, we analyzed another GSH-related enzyme, glyoxalase I,in S.cerevisiae. The enzyme catalyzes detoxification of methylglyoxal with GSH.We cloned the glyoxalase I gene (GLO1) from S.cerevisiae, and investigated its physiological function in yeast cell by analyzing the phenotype of glol-deficiency as well as GLO1-overexpression in the gshl or gsh2 background. We also clarified that expression of the GLO1 gene was regulated by HOG-MAP kinase pathway under highly osmotic conditions.From these situations, we speculated that GSH may be critical for adaptive response to environmental stress in yeast. Less