Systematic Analysis of the Yeast Fatty Acid Desaturase Mutant

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题名:	Systematic Analysis of the Yeast Fatty Acid Desaturase Mutant
作者:	黃稜潔;Huang, Leng-Jie
贡献者:	生命科學系
关键词:	脂肪酸去飽和酶;酵母菌;脂肪酸;磷脂質;fatty acid desaturase;fatty acid;phospholipid;glycerolipid;OLE1;Saccharomyces cerevisiae
日期:	2020-08-20
上传时间:	2020-09-02 15:27:48 (UTC+8)
出版者:	國立中央大學
摘要:	飽和與不飽和脂肪酸之間的平衡是維持細胞膜性質和其他細胞功能的關鍵因素。脂肪酸去飽和酶可催化細胞中不飽和脂肪酸的產生。動物攜帶多種脂肪酸去飽和酶，稱為硬脂酰輔酶A去飽和酶（SCD），而釀酒酵母僅具有一個由OLE1編碼的SCD。該酶對於發芽酵母是維持生命所必需的酵素。然而，目前尚不清楚去飽和酶如何通過控制脂肪酸去飽和來調節細胞功能。在我的研究中，我以系統性分析熱敏感的ole1突變體的表現型來瞭解該問題。首先我使用即時聚合酶鏈鎖反應去偵測OLE1的基因表現，該結果顯示ole1突變體中的OLE1訊息核糖核甘酸高於正常型，這與脂肪酸去飽和度不足會促進OLE1基因表現的觀點一致。藉由環己亞醯胺抑制蛋白質生成而檢驗Ole1降解速率的實驗顯示，Ole1是一種壽命短的蛋白質，突變蛋白質在高溫下更加不穩定，這表明突變可能影響蛋白質折疊並導致不穩定性。脂質組學的分析表明，ole1突變體中不飽和脂肪酸的含量確實降低了，而短鏈脂肪酸的含量卻增加了。此外，ole1突變體中的磷脂酸顯著增加，三酸甘油脂減少。藉由光漂白內質網膜蛋白的螢光並測定其螢光恢復，此實驗顯示ole1突變體的膜流動性降低，表明不飽和脂肪酸有助於維持正常的膜流動性。此外，我進行了全基因組轉錄組分析，以了解脂質變得更飽和時細胞如何反應。該結果顯示鞘脂和固醇生物合成的有關基因表達發生了變化，表明它們與脂質飽和相互作用。總而言之，我的結果揭示了細胞如何藉由全面性的脂質重組和轉錄譜的整體重塑來反應過多的飽和脂肪酸。我的研究開啟一條新的研究方向去了解這些改變如何影響細胞生理。;Balance between saturated and unsaturated fatty acids is a key factor for maintaining the property and function of cellular membranes. The enzyme fatty acid desaturase catalyzes the production of unsaturated fatty acids in cells. While metazoans carry multiple fatty acids desaturases, named Stearoyl CoA Desaturases (SCDs), the budding yeast Saccharomyces cerevisiae has only one SCD, encoded by OLE1. This enzyme is essential for cell viability of budding yeast. However, it is unclear how desaturase regulates cellular processes by controlling fatty acid desaturation. In my study, I attempt to gain insights into this question by systematically analyzing the phenotypes of temperature-sensitive ole1 mutants. I first used the real-time polymerase chain reaction to examine OLE1 gene expression, which showed a higher level of the OLE1 transcript in ole1 mutants than the wild type, consistent with the notion that deficiency in the fatty acid desaturation promotes OLE1 expression. Cycloheximide chase assay showed that Ole1 is a short-lived protein and that the mutant proteins are more unstable at the restrictive temperature, suggesting that the mutations may compromise protein folding and cause instability. The lipidomic analysis showed a reduced level of unsaturated fatty acids and an increase of short chain fatty acids in ole1 mutants. In addition, phosphatidic acid is dramatically increased in ole1 mutants and triacylglyceride is decreased. Fluorescence recovery after photobleaching assay for an endoplasmic reticulum membrane protein showed a reduced mobility in the ole1 mutant, indicating that unsaturated fatty acids contribute to a fluid membrane. Furthermore, I performed genome-wide transcriptome analysis to understand how cells respond to lipid saturation. Notably, the result showed an altered expression profile of genes involved in sphingolipid and sterol biosynthesis, indicating their interplay with lipid saturation. In summary, the results reveal a global remodeling of lipid and transcription profiles in response to lipid saturation. My study opens up new research directions in mechanistic understanding of how these changes impact on cellular physiology.
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