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

本研究的目的是获得一个线索的进化适应性反应的氧化应激酵母。为了实现这一目标，我们对酿酒酵母的几个缺乏抗氧化酶的突变体进行了筛选，并分析了这些突变体细胞对氧化应激的反应。微生物被认为使用细胞色素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）。然而，我们发现了来自酵母菌Hansenula mrakii的GPx。我们克隆了马拉氏血吸虫的类GPx基因，该基因含有一个78 3bp的开放阅读框架。我们还分析了三个GPx同源基因从S.党。GPx活性在所有这些基因的破坏物中均未检测到， 关于我们 其中一个基因的表达通过氧化应激来表达。研究酿酒酵母中GPx的特性将为生物体抗氧化应激机制的进化提供线索，并探讨过氧化氢酶在氧化应激适应性反应中的重要作用。酿酒酵母有两个过氧化氢酶基因（CTT 1和CTA 1），我们破坏了这两个基因。ctt 1 DELTA/cta 1 DELTA双突变体在对数生长期对H_2O_2的敏感性与野生型基本相同，但不能适应H_2O_2胁迫。以前我们报道过gsh 1突变体对H_2O_2高度敏感，对H_2O_2完全不适应。此外，我们已经克隆了OSR 1基因，该基因增强了对脂质过氧化氢引起的氧化应激的抗性。OSR 1过表达细胞内GSH水平升高。综合考虑，过氧化氢酶可能在紧急情况下或作为替代备用发挥作用，而GSH可能在氧化应激反应中发挥更重要的作用。为了证实这一点，我们克隆了编码谷胱甘肽合成酶（谷胱甘肽生物合成的第二个酶）的GSH 2基因。GSH_2 DELTA突变体对氧化胁迫也是高度敏感的，我们进一步阐明了GSH的生物合成在抗氧化胁迫和适应性反应中的重要作用，并研究了GSH的再合成在H_2O_2适应性反应中的作用。GSH被氧化为谷胱甘肽二硫化物（GSSG），并通过谷胱甘肽还原酶（GR）和葡萄糖-6-磷酸脱氢酶（G6 PDH）的作用还原为GSH（还原型）。我们破坏了cach基因，并分析了它们的氧化应激反应表型。GR缺陷型突变体仍能适应H_2O_2胁迫，而G6 PDH缺陷型突变体则不能。G6 PDH提供NADPH，因此，该酶可能不仅为GR提供还原力，而且还在氧化应激以外的其他应激反应中发挥重要作用。本研究克隆了酿酒酵母glycoproteinase I基因（GLO 1），并通过分析glycoproteinase I基因缺陷的表型以及glycoproteinase I基因在gsh 1和gsh 2背景下的过表达，研究了glycoproteinase I基因在酵母细胞中的生理功能。我们还阐明了GLO 1基因在高渗条件下的表达受HOG-MAP激酶途径的调控，由此推测GSH可能是酵母对环境胁迫的适应性反应的关键。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