本文關(guān)鍵詞： 自噬 雷帕霉素 乙�；M學 乙�；� 蛋白質(zhì)翻譯后修飾　出處：《浙江大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的:探究雷帕霉素誘導細胞自噬過程中蛋白質(zhì)乙�；M學的特征。方法:將細胞分為陰性對照組、DMSO對照組、雷帕霉素處理組,采用western blot檢測mTOR以及其底物的磷酸化水平,激光共聚焦觀察內(nèi)源性LC3的熒光點,確立細胞自噬的過程;設(shè)立對照組與0.1μM雷帕霉素處理,采用SILAC標記技術(shù),經(jīng)過胰酶消化、乙酰化賴氨酸抗體富集,并行串聯(lián)質(zhì)譜定量檢測細胞內(nèi)乙�；鞍姿�,而后通過GO、Motif-x、KEGG、CORUM等數(shù)據(jù)庫對乙�；鞍走M行分析。結(jié)果:(1)雷帕霉素處理細胞后,mTOR及其底物RPS6KB1、EIF4G1的磷酸化顯著下調(diào),同時LC3熒光點及LC3-II/LC3-I比值明顯升高,驗證了雷帕霉素可以誘導細胞的自噬。(2)SILAC技術(shù)和質(zhì)譜分析結(jié)果顯示,檢測到944個蛋白上共1808個乙�；稽c,其中397個蛋白上共533個位點乙酰化發(fā)生顯著變化。鑒定出來蛋白中,60.4%僅僅具有一個乙酰化位點,20.4%具有兩個位點,19.2%的蛋白具有多個位點發(fā)生乙�；＿@些乙�；鞍自诩毎|(zhì)、細胞核以及線粒體中均有分布。(3)蛋白基序的分析表明,FxK*、K*xxxxK和KxxxxK*是三個潛在的自噬相關(guān)乙酰化基序。(4)GO富集分析的結(jié)果顯示,大分子復(fù)合體的組裝、細胞代謝過程、基因表達、N端乙酰轉(zhuǎn)移酶活性等相關(guān)的過程均發(fā)生了顯著的乙�；患�(5)KEGG分析發(fā)現(xiàn),與乙酰輔酶A相關(guān)的三大物質(zhì)代謝途徑:糖酵解、脂肪酸代謝與氨基酸代謝過程中大部分的酶受到乙酰化或去乙�；{(diào)控。(6)細胞自噬過程中蛋白質(zhì)修飾以剪切體、核糖體和蛋白酶體等蛋白復(fù)合體的形式出現(xiàn)。(7)乙�；窴AT7在K155位點、EP300的多個位點如K1203、K1542、K1546、K1707均有顯著的乙酰化水平變化,與此對應(yīng)的底物核內(nèi)組蛋白H3的K19、K24,H4K16也發(fā)生了乙酰化變化。結(jié)論:蛋白質(zhì)的乙酰化修飾也是參與細胞自噬的一種重要的蛋白質(zhì)翻譯后修飾方式,且這些蛋白涉及到細胞內(nèi)重要的生物過程,即:轉(zhuǎn)錄依賴途徑和相關(guān)物質(zhì)代謝等轉(zhuǎn)錄非依賴途徑。細胞自噬與乙酰輔酶A相關(guān)代謝途徑的乙酰化調(diào)控密不可分,從側(cè)面印證了乙酰輔酶A是介導雷帕霉素誘導細胞自噬的一個重要的分子靶標。乙酰化酶KAT7和EP300的自我乙�；揎検羌毎允蛇^程中蛋白質(zhì)乙�；揎椀氖种匾姆绞街�,且這兩種酶是參與細胞自噬調(diào)控重要的乙�；D(zhuǎn)移酶。
[Abstract]:Objective: to investigate the characteristics of protein-acetylation in the process of rapamycin induced autophagy. Methods: the cells were divided into negative control group and rapamycin treated group. Western blot was used to detect the phosphorylation level of mTOR and its substrate. The fluorescence point of endogenous LC3 was observed by confocal laser, and the process of autophagy was established. The control group was treated with 0.1 渭 M rapamycin, SILAC labeling technique was used, trypsin was digested and acetyllysine antibody was enriched. The level of acetylated protein in cells was detected by tandem mass spectrometry. The phosphorylation of acetylated protein was analyzed by Gogotif-xKEGGGrum and other databases. Results the phosphorylation of mTOR and its substrate RPS6KB1 EIF4G1 were significantly down-regulated after treated with rapamycin. At the same time, the fluorescence point of LC3 and the ratio of LC3-II/LC3-I were significantly increased, which confirmed that rapamycin could induce autophagy and siliac analysis. The results of mass spectrometry showed that 1 808 acetylation sites of 944 proteins were detected. There were significant changes in acetylation of 533 sites on 397 proteins. 60.4% of the identified proteins had only one acetylated site (20.4%) and two loci (19.2%) had multiple acetylation sites, and these acetylated proteins were found in cytoplasm. The analysis of the protein motifs in the nucleus and mitochondria showed that FxKOXXXK and KxxxxK* were three potential autophagy associated acetylated motifs. The results of enrichment analysis showed that the assembly of macromolecular complexes and the process of cell metabolism. KEGG analysis showed that there were three major metabolic pathways related to acetylcoenzyme A: glycolysis, which were related to the activity of N-terminal acetyltransferase, and the results of KEGG analysis showed that there were three major metabolic pathways related to acetyl coenzyme A, such as glycolysis, glycolysis, glycolysis, glycolysis and glycolysis. During fatty acid metabolism and amino acid metabolism, most of the enzymes were modified by proteins during autophagy, which were regulated by acetylation or deacetylation. In the form of ribosome and proteasome protein complexes, the acetylase KAT7 had significant changes in acetylation level at several sites of K155 EP300, such as K1203, K1542, K1546 and K1707. The acetylation of the corresponding histone H3 K19 K24H4K16 also occurred. Conclusion: the acetylation modification of proteins is also an important post-translational modification of proteins involved in autophagy. These proteins are involved in important biological processes in cells, that is, transcription dependent pathway and related substance metabolism. Autophagy is closely related to acetylation regulation of acetylcoenzyme A related metabolic pathway. It is proved that acetyl coenzyme A is an important molecular target to mediate rapamycin induced autophagy. The self-acetylation modification of acetylases KAT7 and EP300 is one of the most important ways to modify protein acetylation during autophagy. These two enzymes are important acetyltransferases involved in the regulation of autophagy.
【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R96

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