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

本研究的目的是获得酵母对氧化应激适应性反应的进化线索。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体，并分析了这些突变体细胞对氧化应激的反应。人们一直认为微生物不具有以谷胱甘肽（GSH）为电子供体的过氧化物酶。微生物被认为使用细胞色素c作为过氧化物酶反应（细胞色素c过氧化物酶）的电子供体。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，谷胱甘肽被用作电子供体（谷胱甘肽过氧化物酶，GPx）。然而，我们从酵母中发现了一个GPx， Hansenula mrakii。我们从mraki中克隆了GPL1（gpx样）基因，该基因包含一个783bp的开放阅读框。我们还分析了来自酿酒酵母的三个gpx同源基因。所有这些基因的干扰物中均未检测到GPx活性，氧化应激表达了其中一个基因的更多表达。GPx在S.cerevisiae中的表征将为生物体抗氧化应激机制的进化提供线索。我们还研究了过氧化氢酶在氧化应激适应性反应中的重要性。酿酒酵母有两个过氧化氢酶基因（CTT1和CTA1），我们破坏了这两个基因。ctt1 DELTA/cta1DELTA双干扰物在log期对H_2O_2的敏感性与野生型细胞基本相同，但该突变体对H_2O_2胁迫表现出不适应。在此之前，我们报道了gsh1突变体对H_2O_2敏感，完全不能适应H_2O_2。此外，我们还克隆了提高脂质过氧化氢氧化应激抗性的OSR1基因。osr1过表达细胞内GSH水平升高。综上所述，过氧化氢酶可能在紧急情况下发挥作用，或者作为一种替代备用，而谷胱甘肽可能在氧化应激反应中发挥更重要的作用。为了验证这一点，我们克隆了编码谷胱甘肽合成酶的GSH2基因，谷胱甘肽合成酶是谷胱甘肽生物合成的第二种酶。gsh2 DELTA突变体也对氧化应激敏感。研究结果表明，GSH的生物合成对植物抗氧化和适应氧化应激具有重要作用，并进一步研究了GSH回收对H_2O_2适应性的影响。谷胱甘肽被氧化为谷胱甘肽二硫（GSSG），并通过谷胱甘肽还原酶（GR）和葡萄糖-6-磷酸脱氢酶（G6PDH）的作用还原为谷胱甘肽（还原形式）。我们破坏了每个基因，并分析了它们与氧化应激反应有关的表型。g6pdh缺陷突变体对H_2O_2的适应能力较弱，而gr缺陷突变体对H_2O_2的适应能力较弱。G6PDH提供NADPH，因此，G6PDH酶可能在GR中发挥一定作用，除了提供还原能力外，还可能在氧化应激的其他应激反应中发挥重要作用。除了这些酶外，我们还分析了酿酒酵母中另一种与谷胱甘肽相关的酶，乙醛酶I。该酶用谷胱甘肽催化甲基乙二醛解毒。从酿酒酵母中克隆了glyoxalase I基因（GLO1），并通过分析gshl和gsh2背景下GLO1-过表达和gl1 -缺乏表型，研究了该基因在酵母细胞中的生理功能。我们还澄清了在高渗透条件下GLO1基因的表达受HOG-MAP激酶途径的调控。从这些情况来看，我们推测谷胱甘肽可能对酵母对环境胁迫的适应性反应至关重要。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