未知功能DUF28 protein及色胺酸調控途徑在酵母菌氧化逆境反應之功能分析

Functional study of DUF28 protein and tryptophan-related metabolism in Saccharomyces cerevisiae response to oxidative stress

陳巧燕(Chiao-Yen Chen)

指導教授 ： 鄭秋萍

國立臺灣大學/生命科學院/植物科學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.00220

摘要

生物在正常生長代謝過程中，會產生活性氧化物質(reactive oxygen species, ROS)，做為訊息傳導分子，幫助生物抵抗逆境，順利進行生長發育。適量的ROS可以保護生物，但過量的ROS反而會破壞蛋白質、脂質及DNA，造成細胞損壞，導致細胞死亡。本論文針對酵母菌S. cerevisia兩個基因，利用遺傳、分子及DNA微陣列等研究策略，分析它們在氧化逆境的功能進行研究。首先，YGR021w產物係隸屬具跨物種保守性但功能未知的新穎DUF28蛋白家族；本論文的研究結果發現，此基因在不同氧化逆境下其表現會被抑制，而其剔除株對於氧化逆境的抗性增加，且提早或大量誘導具代表性之已知抗氧化逆境相關基因的表現，而過量表現株的特質則反之；故首次發現DUF28蛋白在酵母菌抗氧化逆境機制中具負面調控。另外，微陣列結果進一步顯示，在YGR021w剔除株中抗逆境相關基因較提早且大量表現，醣類及能量相關基因也大量表現，可能在醣類及能量上能更有效之利用，且其細胞內之氧化還原之平衡，也較WT更具穩定性，顯示YGR021w剔除菌株之耐受性，可能是多種有利因素而得到之結果。其次，本論文也以色胺酸合成酶YGL026c (TRP5)為研究對象，結果發現，此基因在不同氧化逆境下其表現會被誘導，TRP5剔除株對過氧化氫及膜相關逆境之敏感度增加，且在氧化逆境下TRP5剔除株中與抗氧化逆境相關之基因的誘導表現比在WT中較為延遲及減弱。另外，微陣列結果顯示，在正常條件下，TRP5缺失即會造成酵母菌本身與膜構造及抗逆境相關基因之表現下降；而在氧化逆境下，其抗氧化、抗其他逆境及蛋白質降解相關基因之表現也是下降，可能在TRP5剔除株中大量之ROS造成之損壞物質無法藉由蛋白質降解過程移除，且缺乏了移除危機之能力，這些皆可能為造成TRP5剔除菌株對氧化逆境敏感的原因。這些結果顯示，酵母菌在遭受逆境時，需有具正負調控能力的不同的蛋白共同參與並協調，以達到最佳的最後逆境反應結果，以維持其生存。

關鍵字

酵母菌 ； 氧化逆境 ； DUF28蛋白 ； 色胺酸

並列摘要

Cells growing aerobically are exposed to reactive oxygen species (ROS) generated during metabolism. These ROS can seriously damage the cell by reacting with cellular components, causing oxidative stress and cell death. The aim of this study is to investigate roles of two S. cerevisiae genes, YGR021w and YGL026c (tryptophan synthase, TRP5), in oxidative stress response by taking genetic, molecular and microarray approaches. Firstly, YGR021w is predicted to encode a protein belonging to a conserved novel protein family, namely DUF28. The results showed: (1) YGR021w expression was repressed by oxidative stress factors; (2) Deletion of YGR021w led to enhanced tolerance to hydrogen peroxide and heat shock stress, and YGR021w over-expression had opposite effects; (3) Stress-induced expression of oxidative response-related genes in YGR021w-deleted strain was faster and stronger compared to the wild-type strain (WT) ; (4) Microarray-based transcriptome analysis revealed enhanced induction of genes involved in oxidative response, carbohydrate metabolism in YGR021w-deleted strain compared to the WT. As the first report, these results together indicated that YGR021w plays a negative role in yeast response to oxidative stress. Secondly, role of TRP5 in oxidative stress has not been elucidated until this study. The results showed: (1) TRP5 expression was induced by oxidative stress factors; (2) Deletion of the TRP5 led to remarkably increased sensitivity to various oxidative stresses and hydrophobic toxic compounds, while complementation rescued some of these defects; (3) Stress-induced expression of oxidative response-related genes in TRP5-deleted strain was much reduced compared to the WT; (4) Exogenous supplement of plant hormone IAA, enhanced tolerance to oxidative stress in the WT and the TRP5-deleted strain; (5) Microarray analysis revealed decreased expression of genes involved in stress tolerance and protein degradation in the deleted strain compared to the WT. Therefore, in addition to possible loss of cell structure integrity, the accumulation of abnormal proteins under stress condition in TRP5-deleted strain, which is not capable of degrading and removing such proteins, may account for the increased sensitivity of TRP5-deleted strain to oxidative stress. These results pointed out that TRP5 plays an important role in yeast stress response. Taken together, the results suggest that both negative and positive elements are required to involve and coordinate to achieve proper and efficient response in yeast to oxidative stress.

並列關鍵字

S. cerevisiae ； oxidative stress ； DUF28 ； tryptophan

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