高温和氧化条件下酵母细胞应激产生活性物质的研究
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摘要
本论文对酵母细胞在高温和氧化条件下的应激反应进行了研究，通过低剂量刺激酵母细胞产生活性衍生物—LYCD，并筛选了耐高温酵母突变株，开辟了制备LYCD的新途径。
本课题采用呼吸缺陷和正常的酵母菌，分析线粒体功能在酵母氧化应激和热应激反应中的作用。结果发现，呼吸缺陷酵母抵抗致死剂量氧化剂的能力比正常酵母差，说明线粒体对于细胞抗氧化是必需的。而呼吸缺陷酵母的抗热能力却比正常酵母强，说明线粒体对于细胞抗热具有阻碍作用。通过低剂量的预处理，两种酵母都对相应致死剂量产生适应性，说明适应性的产生与线粒体功能无关。
测定了酵母预处理前后细胞内几种重要抗氧化剂(GSH、SOD、CAT)的变化，结果表明：两种预处理都可以使正常酵母细胞中抗氧化剂含量提高。并分析了正常酵母预处理前后细胞内脂质过氧化物—丙二醛(MDA)的含量，发现两种预处理后，再在致死条件下作用，MDA的含量降低，细胞的存活率也升高。这不仅说明，预处理使细胞抗氧化水平提高，减少了MDA的积累，细胞增加了对氧化和热的抗性，更直接说明热致死与氧化关系密切。
将经过H_2O_2和37℃预处理后的酵母进行破壁，分别得到LYCD_1和LYCD_2，发现两者都对细胞抗氧化具有保护活性。分别对其处理条件进行优化，确定了LYCD_1的最佳处理条件为：0.4mmol／L H_2O_2预处理60min，LYCD_2的最佳处理条件为：37℃预处理30min。
通过紫外诱变筛选了耐高温酵母突变株，发现与正常酵母相比，耐高温突变株对热和氧化都具有很强的抗性。而且耐高温酵母提取物也具有保护细胞抵抗氧化的能力。并分析了正常酵母和耐高温酵母细胞内GSH、SOD、CAT的差异，发现耐高温酵母胞内抗氧化水平高于正常酵母菌株。
对正常酵母预处理前后蛋白的变化进行了双向电泳分析，结果表明，有一些蛋白是H_2O_2和37℃都可以诱导合成的，但也有些蛋白是H_2O_2或37℃特异性诱导合成的。此外，对耐高温酵母的蛋白分析后，发现它与正常酵母相比，产生了很多新的蛋白，其中包括热激蛋白，也应包括与细胞抗氧化有关的蛋白。
This study was based on stress response of yeast cell under high temperature and oxidative condition. Live Yeast Cell Derivative (LYCD) was produced by yeast cell, which was pretreated by low dose temperature and oxidative condition. The experiment setup the new method producing LYCD by selecting thermotolerant mutant.
With comparative the stress response of yeast and respiratory-deficient strains, the effect of mitochondria under oxidative stress and heat stress were analyzed. Our results indicated that mitochondria! function was required for resistance to oxidative stress in yeast, since respiratory-deficient strains had the less survival than the yeast. But respiratory-deficient strains had the more ability for resistance to high temperature, indicating that it was inhibited by mitochondrial function to resist heat stress. Through pretreating by low dose, yeast and respiratory-deficient strains were able to mount an adaptive response to high temperature and H2O2.It showed that mitochondrial was unrequired in adaptive response of yeast.
The following conclusions were drawn by testing the change of antioxidants and MDA in yeast cell: pretreating of low dose could increase the content of GSH and the activities of SOD and CAT, could reduce the accumulation of MDA. And these induced the resistance to lethal concentration of 50 and H2O2. Furthermore study showed that heat and oxidative stress may have a common mechanism of damage.
. LYCD1 and LYCD2 were produced by breaking up the yeast cell pretreated with H2O2 and 37 C. The results showed that LYCD1 and LYCD2 could protect cell to resist oxidative condition. The optimum conditions of producing LYCD1 and LYCD2 were as follow: the concentration of H2O2 is 0.4 mmol/L, and the treating time is 60min;the temperature is 37 C,and the treating time is 30min.
After UV treating, the thermotolerant mutant was selected. And it had the more ability for resistance to high temperature and oxidative condition. The extract of mutant had biological activities, the content of GSH and the activities of SOD and CAT in mutant were higher than the initial strain.
Through two-dimensional gel electrophoresis experiment, the proteins were analysed. Some changes were induced by H2O2 and 37 C,which were not made in untreated cells. Other changes were induced by H2O2 or 37 C. hi addition, the proteins of mutant were analysed. The result indicated that lots of new proteins were synthesized, some were heat-shock proteins and others were relative to anti-oxidative.
本课题采用呼吸缺陷和正常的酵母菌，分析线粒体功能在酵母氧化应激和热应激反应中的作用。结果发现，呼吸缺陷酵母抵抗致死剂量氧化剂的能力比正常酵母差，说明线粒体对于细胞抗氧化是必需的。而呼吸缺陷酵母的抗热能力却比正常酵母强，说明线粒体对于细胞抗热具有阻碍作用。通过低剂量的预处理，两种酵母都对相应致死剂量产生适应性，说明适应性的产生与线粒体功能无关。
测定了酵母预处理前后细胞内几种重要抗氧化剂(GSH、SOD、CAT)的变化，结果表明：两种预处理都可以使正常酵母细胞中抗氧化剂含量提高。并分析了正常酵母预处理前后细胞内脂质过氧化物—丙二醛(MDA)的含量，发现两种预处理后，再在致死条件下作用，MDA的含量降低，细胞的存活率也升高。这不仅说明，预处理使细胞抗氧化水平提高，减少了MDA的积累，细胞增加了对氧化和热的抗性，更直接说明热致死与氧化关系密切。
将经过H_2O_2和37℃预处理后的酵母进行破壁，分别得到LYCD_1和LYCD_2，发现两者都对细胞抗氧化具有保护活性。分别对其处理条件进行优化，确定了LYCD_1的最佳处理条件为：0.4mmol／L H_2O_2预处理60min，LYCD_2的最佳处理条件为：37℃预处理30min。
通过紫外诱变筛选了耐高温酵母突变株，发现与正常酵母相比，耐高温突变株对热和氧化都具有很强的抗性。而且耐高温酵母提取物也具有保护细胞抵抗氧化的能力。并分析了正常酵母和耐高温酵母细胞内GSH、SOD、CAT的差异，发现耐高温酵母胞内抗氧化水平高于正常酵母菌株。
对正常酵母预处理前后蛋白的变化进行了双向电泳分析，结果表明，有一些蛋白是H_2O_2和37℃都可以诱导合成的，但也有些蛋白是H_2O_2或37℃特异性诱导合成的。此外，对耐高温酵母的蛋白分析后，发现它与正常酵母相比，产生了很多新的蛋白，其中包括热激蛋白，也应包括与细胞抗氧化有关的蛋白。
This study was based on stress response of yeast cell under high temperature and oxidative condition. Live Yeast Cell Derivative (LYCD) was produced by yeast cell, which was pretreated by low dose temperature and oxidative condition. The experiment setup the new method producing LYCD by selecting thermotolerant mutant.
With comparative the stress response of yeast and respiratory-deficient strains, the effect of mitochondria under oxidative stress and heat stress were analyzed. Our results indicated that mitochondria! function was required for resistance to oxidative stress in yeast, since respiratory-deficient strains had the less survival than the yeast. But respiratory-deficient strains had the more ability for resistance to high temperature, indicating that it was inhibited by mitochondrial function to resist heat stress. Through pretreating by low dose, yeast and respiratory-deficient strains were able to mount an adaptive response to high temperature and H2O2.It showed that mitochondrial was unrequired in adaptive response of yeast.
The following conclusions were drawn by testing the change of antioxidants and MDA in yeast cell: pretreating of low dose could increase the content of GSH and the activities of SOD and CAT, could reduce the accumulation of MDA. And these induced the resistance to lethal concentration of 50 and H2O2. Furthermore study showed that heat and oxidative stress may have a common mechanism of damage.
. LYCD1 and LYCD2 were produced by breaking up the yeast cell pretreated with H2O2 and 37 C. The results showed that LYCD1 and LYCD2 could protect cell to resist oxidative condition. The optimum conditions of producing LYCD1 and LYCD2 were as follow: the concentration of H2O2 is 0.4 mmol/L, and the treating time is 60min;the temperature is 37 C,and the treating time is 30min.
After UV treating, the thermotolerant mutant was selected. And it had the more ability for resistance to high temperature and oxidative condition. The extract of mutant had biological activities, the content of GSH and the activities of SOD and CAT in mutant were higher than the initial strain.
Through two-dimensional gel electrophoresis experiment, the proteins were analysed. Some changes were induced by H2O2 and 37 C,which were not made in untreated cells. Other changes were induced by H2O2 or 37 C. hi addition, the proteins of mutant were analysed. The result indicated that lots of new proteins were synthesized, some were heat-shock proteins and others were relative to anti-oxidative.
引文
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[6] 李芳红．线粒体DNA的氧化损伤[J]．国外医学卫生学分册，2000，27(1)，33-36
[7] Kanner J, German JB. Initition of lipid peroxidation in biological systems. CRC Crit Rev Food Sci Nrtr,25,1997,317-325
[8] Scandalios. Response of plant antioxidant defense genes to environment stress [J]. Advance in Genetics,1990,28:2-35.
[9] Christman, M.F.Morgan,R.W. Positive control of a regulon for defense against oxidative stress and some heat shock protein in Salmondla typhimuriumm [J]. Cell, 1985,41:753-7626
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[12] Marcos DDP, and Anita D. Panc. Acquisition of tolerance against oxidative damage in S.cerevisiae [J]. Microbiology,2001,2:1-11
[13] Jin Sook Jeong,Soo Jin Kwon, Sang Won Kang, Sue Goo Rhee, and Kanghua Kim. Purification and characterization of a second Type Thioredoox Peroiddase from S.cerevisiae [J]. Biochemistry ,1999,38:776-783
[14] Angela M.Avery and Simon V.Avery. JBC S.cerevisiae Expresses Three Phospholipid Hydroperoxide Glutathione peroxidases. Papers in press .July 9,2001 M105672200
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[21] Muller, E. G. D. A glutathione reductase mutant of yeast accumulates high levels of oxidized glutathione and requires thiredoxin for growth. [J]. Mol. Biol. Cell. 1996, 7, 1895-1813
[22] Luikenhuis,S., Perrone, G, Dawes, L. W. The yeast S.cerevisiae contains two glutaredoxin genes that are required for protection against reactive oxygen species. [J]. Mol. Biol. Cell, 1998, 9, 1081-1091
[23] Rikhvanov, E.G. Heat shock-induced changes in the respiration of the yeast S.cerevisiae.[J].Microbiology.2001, 70, 531-535
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[26] Moye_Rowley, W. S., Harshman, K. D., and Parker, C. S. Yeast Yapl, encodes a novel form of the jun family of transcriptional activator proteins [J]. Genes Dev,.1989,3: 283-292
[27] Hofman-Baang.Nitrogen catabolite repression in S.cerevisiae [J]. Mol Biotechmol, 1999,12:35-73
[28] Cheng L. Watt, R., and Piper, P.W. Polyubiquitin gene expression contributes to oxidative stress resistance in respiratory yeast [J]. Mol Gen Genet, 1994,243:358-362
[29] Hirata, d.,Yano,K and Miyakawa, T. Stress-induceddtraneriptional activaation mediated by YAP landYAP2genes that encode the Jun family of transcriptional activators in S.cerevisiae.[J]. Mol.Gen.Genet, 1994,242:250-256
[30] Engelberg,D.,et al.The UV responseinvolving the RAS signaling pathway and AP-1 transcrition ffactors is conserved between yeast and mammals. [J] . Cell, 1994,77:381-390
[31] Jungmann,J.,et al.MACl, a nuclear regulatory protein related to Cu-dependent transcription factors and is involved in Cu/Fe utilization and stress resistance in yeast[J]. EMBO, 1993,12:5051-5056
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[34] Jerold Z.Kaplan.M .D. Acceleration of wound healing by a live yeast cell derivative [J]. Arch Surg, 1984, 119:1005-1008
[35] Willem H. Mager, Pedro moradas Ferreim. Stress response of yeast. [J]. Biochem, 1993, 290, 1-13
[36] Marchler, G., Schuller, C., Adam, G. A S. cerevisiae UAS element controlled by protein kinase A activates transcription in response to a variety of stress conditions. [J]. EMBO J. 1993, 12, 1997-2003
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[2] 赵克然，杨毅军，曹道俊．氧自由基与临床[M]．中国医药科技出版社，2000，1-56
[3] 孙存普．自由基生物学导论[M]．中国科技大学出版社，1999，66-81
[4] 赵保路．氧自由基和天然抗氧剂[M]．北京：科学出版社，1999，1-20．
[5] 薛丽君，金中初．真核细胞氧化应激及其适应机制[J]．国外医学卫生学分册，1997，24(4)，207-210
[6] 李芳红．线粒体DNA的氧化损伤[J]．国外医学卫生学分册，2000，27(1)，33-36
[7] Kanner J, German JB. Initition of lipid peroxidation in biological systems. CRC Crit Rev Food Sci Nrtr,25,1997,317-325
[8] Scandalios. Response of plant antioxidant defense genes to environment stress [J]. Advance in Genetics,1990,28:2-35.
[9] Christman, M.F.Morgan,R.W. Positive control of a regulon for defense against oxidative stress and some heat shock protein in Salmondla typhimuriumm [J]. Cell, 1985,41:753-7626
[10] Cohen, G., Rapatz, W. And Ruis, G. Sequence of the S.cerevisiae CTA1 gene and amino acid sequence of catalase A derived from it [J]. Gen. Microbiol , 1988,176,159-163
[11] Hartig, A. And Ruis, H. Nucleotide sequence of the S.cerevisiae CTT1 gene and deduced amino-acid sequence of yeast catalase T [J]. Eur.J.Biochem, 1986,160:487-490
[12] Marcos DDP, and Anita D. Panc. Acquisition of tolerance against oxidative damage in S.cerevisiae [J]. Microbiology,2001,2:1-11
[13] Jin Sook Jeong,Soo Jin Kwon, Sang Won Kang, Sue Goo Rhee, and Kanghua Kim. Purification and characterization of a second Type Thioredoox Peroiddase from S.cerevisiae [J]. Biochemistry ,1999,38:776-783
[14] Angela M.Avery and Simon V.Avery. JBC S.cerevisiae Expresses Three Phospholipid Hydroperoxide Glutathione peroxidases. Papers in press .July 9,2001 M105672200
[15] Marchler, G.,et al. A S.cerevisiae USAelement controlled by protein kinase A activates transcription in response to a variety of stress conditions [J]. EMBO,1993,12:1997-2003
[16] 贾耀勇．人类健康与美的使者——金属硫蛋白[J]．北京日化，2001，3(64)，15-18
[17] Culotta, V.C., Howard, W.R. and Liu, X. F. CRS5 encodes a metallothionein-lide
protein in S.cerevisiae[J]. Biol.Chem. 269, 25295-25302
[18] Tamai,K.T., Gralla, E.B., Ellerby, L.M. Yeast and mammalian metallothions functionally substitute for yeast copper-zinc superoxide[J]. Proc. Natl Acad. Sci, USA, 1993, 90,8013-8017
[19] Zhao,X.J., Raitt, D., Burke, P.V. Function and expression of flavohemoglobin in S. cerevisiae, [J]. Biol. Chem. 1996,271,25131-25138
[20] Muller, E. G. D. Thioredoxin deficiency in yeast prolongs S phase and shortens the G1 interval of the cell cycle [J]. Bio.Chem.1991, 266, 9194-9202
[21] Muller, E. G. D. A glutathione reductase mutant of yeast accumulates high levels of oxidized glutathione and requires thiredoxin for growth. [J]. Mol. Biol. Cell. 1996, 7, 1895-1813
[22] Luikenhuis,S., Perrone, G, Dawes, L. W. The yeast S.cerevisiae contains two glutaredoxin genes that are required for protection against reactive oxygen species. [J]. Mol. Biol. Cell, 1998, 9, 1081-1091
[23] Rikhvanov, E.G. Heat shock-induced changes in the respiration of the yeast S.cerevisiae.[J].Microbiology.2001, 70, 531-535
[24] John E Davison. Oxidative stress is involved in heat-induced cell death in S.cerevisiae, [J] .Proc.Natl.Acad.Sci.USA. 1995, 93, 5116-5121
[25] Femandes, L.,Rodrigrigues-Pousada, C, and Struhl, K. Yap, a novel family of eight bzip proteins in S.cerevisiae [J]. Mol Cell Biol, 1997,17:6982-6993
[26] Moye_Rowley, W. S., Harshman, K. D., and Parker, C. S. Yeast Yapl, encodes a novel form of the jun family of transcriptional activator proteins [J]. Genes Dev,.1989,3: 283-292
[27] Hofman-Baang.Nitrogen catabolite repression in S.cerevisiae [J]. Mol Biotechmol, 1999,12:35-73
[28] Cheng L. Watt, R., and Piper, P.W. Polyubiquitin gene expression contributes to oxidative stress resistance in respiratory yeast [J]. Mol Gen Genet, 1994,243:358-362
[29] Hirata, d.,Yano,K and Miyakawa, T. Stress-induceddtraneriptional activaation mediated by YAP landYAP2genes that encode the Jun family of transcriptional activators in S.cerevisiae.[J]. Mol.Gen.Genet, 1994,242:250-256
[30] Engelberg,D.,et al.The UV responseinvolving the RAS signaling pathway and AP-1 transcrition ffactors is conserved between yeast and mammals. [J] . Cell, 1994,77:381-390
[31] Jungmann,J.,et al.MACl, a nuclear regulatory protein related to Cu-dependent transcription factors and is involved in Cu/Fe utilization and stress resistance in yeast[J]. EMBO, 1993,12:5051-5056
[32] Zitomer, R.S.and Lowry, C.V.Regulation of gene expression by oxygen in S.cerevisiae [J]. Microbiol.Rev, 1992,56:1-11
[33] Martinez-Pastor, M.T., et al The S.cerevisiae zinc fingere proteins Msnlpand Msn4p are required for transcriptional induction through the stress-response edement [J]. EMBO, 1996,15:2227-2235
[34] Jerold Z.Kaplan.M .D. Acceleration of wound healing by a live yeast cell derivative [J]. Arch Surg, 1984, 119:1005-1008
[35] Willem H. Mager, Pedro moradas Ferreim. Stress response of yeast. [J]. Biochem, 1993, 290, 1-13
[36] Marchler, G., Schuller, C., Adam, G. A S. cerevisiae UAS element controlled by protein kinase A activates transcription in response to a variety of stress conditions. [J]. EMBO J. 1993, 12, 1997-2003
[37] Praekelt, U. M. and Meacock, P. A. HSP12, a new small heat shock gene of S.cerevisiae: analysis of structure, regulation and function. [J]. Mol. Gen. Genet. 1990,223,97-106
[38] Werner-Washburne, M., Becker, J. Yeast Hsp70 RNA levels vary in response to the physiologial status of the cell. [J]. Bacteriol, 1989,171, 2680-2688
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