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

酵母抗活性氧应激适应机制的分子进化

基本信息

批准号：
08456053
负责人：
KIMURA Akira
金额：
$ 3.84万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08456053/
关键词：
Saccharomyces cerevisiae Hansenula mrakii glutathione glutathione peroxidase glutathione reductase glutathione synthetase catalase MAP kinase Hansenula mrakii catalase yAP-1

项目摘要

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

本研究的目的是获得酵母对氧化应激适应性反应进化的线索。为了实现这一目标，我们构建了几种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如何响应氧化应激。人们一直认为，微生物不具有电子供体为谷胱甘肽（GSH）的过氧化物酶。据信微生物使用细胞色素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 用作电子供体（谷胱甘肽过氧化物酶，GPx）。然而，我们从酵母 Hansenula mrakii 中发现了 GPx。我们从H.mrakii中克隆了GPL1(GPx-like)基因，该基因包含一个78 3bp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。在所有这些基因的破坏体中均未检测到 GPx 活性，并且其中一个基因的表达是通过氧化应激表达的。酿酒酵母中GPx的表征将为我们了解生物体中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双突变体在对数期对H_2O_2的敏感性与野生型细胞几乎相同，但该突变体不能表现出对H_2O_2胁迫的适应。此前，我们报道了gsh1突变体对H_2O_2高度敏感，并且根本不能适应H_2O_2。此外，我们还克隆了 OSR1 基因，该基因增强了对脂质过氧化氢引起的氧化应激的抵抗力。 OSR1 过表达细胞中细胞内 GSH 水平增加。总而言之，过氧化氢酶可能在紧急情况下发挥作用或作为替代品，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种酶）的 GSH2 基因。 gsh2 DELTA 突变体对氧化应激也高度敏感。如上所述，我们清楚地认识到，GSH 的生物合成对于抗性以及对氧化应激的适应性反应非常重要，然后我们研究了 GSH 循环对 H_2O_2 适应性反应的影响。 GSH 被氧化为谷胱甘肽二硫化物 (GSSG)，并在谷胱甘肽还原酶 (GR) 和葡萄糖-6-磷酸脱氢酶 (G6PDH) 的作用下还原为还原型 GSH（还原型）。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。 GR缺陷型突变体仍能表现出对H_2O_2胁迫的适应，而G6PDH缺陷型突变体则不能。 G6PDH提供NADPH，因此，该酶不仅可以为GR提供还原能力，而且还可以在氧化应激以外的其他应激反应中发挥关键作用的其他酶发挥一定的作用。除了这些酶之外，我们还分析了酿酒酵母中的另一种GSH相关酶，即乙二醛酶I。该酶通过谷胱甘肽催化甲基乙二醛解毒。我们从酿酒酵母中克隆了乙二醛酶I基因(GLO1)，并通过分析gshl或gsh2背景下gol缺乏和GLO1过表达的表型来研究其在酵母细胞中的生理功能。我们还阐明，在高渗透压条件下，GLO1基因的表达受到HOG-MAP激酶途径的调节。从这些情况，我们推测GSH可能对于酵母对环境胁迫的适应性反应至关重要。较少的