IZAWA Shingo Kyoto Univ., Research Institute for Food Science, Instructor, 食糧科学研究所, 助手 (10273517)
INOUE Yoshiharu Kyoto Univ., Research Institute for Food Science, Associate Professor, 食糧科学研究所, 助教授 (70203263)
|Budget Amount *help
¥6,000,000 (Direct Cost : ¥6,000,000)
Fiscal Year 1997 : ¥2,300,000 (Direct Cost : ¥2,300,000)
Fiscal Year 1996 : ¥3,700,000 (Direct Cost : ¥3,700,000)
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
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