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標題: | 質譜分析自由基對ATG4B上半胱胺酸的修飾 Mapping the reactive oxygen species mediated cysteine modification patterns on ATG4B |
作者: | Hsien-I Chiu 邱顯壹 |
指導教授: | 楊維元 |
關鍵字: | 細胞自噬,ATG4B,ROS,轉譯後修飾,區分性烷基化標定, autophagy,ATG4B,ROS,PTM,differential alkylation labeling, |
出版年 : | 2011 |
學位: | 碩士 |
摘要: | 細胞自噬(Autophagy)現象是真核生物進行大尺度代謝體內物質的途徑,近年來的研究指出此一維持細胞內部物質平衡的過程與各種疾病與生理過程息息相關,如癌症、神經退化病變、老化、免疫機制、細胞發育與凋亡等等,都有相當多的研究指出細胞自噬的重要性。而此代謝途徑與活性氧分子(Reactive Oxygen Species, ROS)的調控又是密不可分的,然而許多研究都只著重於ROS分子與細胞自噬現象上的關聯,僅有少數研究在尋找受到ROS影響而形成的轉譯後修飾是如何對於各細胞自噬相關蛋白(ATG)造成特性的變化。
先前的研究已經指出ATG4A蛋白可以在體外模擬(in vitro)的情況下單獨以H2O2進行半胱胺酸(cysteine)的氧化還原並且藉此調控其活性,但在其餘ATG4同源蛋白的調控機制仍然不明確的情況下,本研究在嘗試建立以質譜儀分析ATG4B蛋白上因ROS分子所造成的轉譯後修飾,並找出修飾前後對其蛋白水解活性的改變的關聯性。我們先以不同濃度的H2O2模擬ATG4B受到ROS分子調控的情形,發現到ATG4B蛋白活性對於ROS分子的敏感性高過ATG4A,並使用區分性烷基化的方法先標定還原狀態的半胱胺酸,再經過化學還原後標定新生的半胱胺酸,使用質譜儀分析出各半胱胺酸的不同烷基化狀態,得知ATG4B上第78、189號半胱胺酸可能是扮演著感受ROS分子信號並調控ATG4B活性的角色。 Autophagy is a large-scale metabolic pathway to degrade cytosolic substances in eukaryotes, it controls the balance of cytosolic materials. Recent studies pointed out that autophagy is closely related to various diseases and physiological processes such as cancer, neurodegeneration, innate & adaptive immunity, ageing, development and cell death, etc. It has been shown that autophagy and ROS signals are inextricable linked. Many studies emphasized on changes in autophagy induced by ROS, whereas only few studies focused on whether ROS-induced ATG proteins’ post-transcriptional modifications would affect these proteins’ functions. A preliminary study showed that ATG4A could be oxidized by H2O2 and lost its enzyme activity, But, it could be reduced and regain its function. Since the regulatory mechanism of other ATG4 family proteins is still unclear, we attempt to establish a MS based analysis method to monitor ROS-mediated post-transcriptional modifications on ATG4B, and further elucidate the relationships between these PTM and ATG4B’s activity. We first treated ATG4B with different concentration of H2O2 to simulate ROS-mediated Cys oxidation and found that ATG4B is more sensitive to ROS than ATG4A. Also, we perform differential alkylation labeling to demarcate various oxidation states of ATG4B’s Cys. The analysis by mass spectrometry suggests that Cys 78 and Cys 189 on ATG4B are the ROS sensor which regulate ATG4B’s activity. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/40220 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科學研究所 |
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