雷帕霉素诱导细胞自噬过程中蛋白质乙酰化组学特征的分析

发布时间：2018-02-24 08:09

本文关键词： 自噬雷帕霉素乙酰化组学乙酰化酶蛋白质翻译后修饰　出处：《浙江大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:探究雷帕霉素诱导细胞自噬过程中蛋白质乙酰化组学的特征。方法:将细胞分为阴性对照组、DMSO对照组、雷帕霉素处理组,采用western blot检测mTOR以及其底物的磷酸化水平,激光共聚焦观察内源性LC3的荧光点,确立细胞自噬的过程;设立对照组与0.1μM雷帕霉素处理,采用SILAC标记技术,经过胰酶消化、乙酰化赖氨酸抗体富集,并行串联质谱定量检测细胞内乙酰化蛋白水平,而后通过GO、Motif-x、KEGG、CORUM等数据库对乙酰化蛋白进行分析。结果:(1)雷帕霉素处理细胞后,mTOR及其底物RPS6KB1、EIF4G1的磷酸化显著下调,同时LC3荧光点及LC3-II/LC3-I比值明显升高,验证了雷帕霉素可以诱导细胞的自噬。(2)SILAC技术和质谱分析结果显示,检测到944个蛋白上共1808个乙酰化位点,其中397个蛋白上共533个位点乙酰化发生显著变化。鉴定出来蛋白中,60.4%仅仅具有一个乙酰化位点,20.4%具有两个位点,19.2%的蛋白具有多个位点发生乙酰化。这些乙酰化蛋白在细胞质、细胞核以及线粒体中均有分布。(3)蛋白基序的分析表明,FxK*、K*xxxxK和KxxxxK*是三个潜在的自噬相关乙酰化基序。(4)GO富集分析的结果显示,大分子复合体的组装、细胞代谢过程、基因表达、N端乙酰转移酶活性等相关的过程均发生了显著的乙酰化富集。(5)KEGG分析发现,与乙酰辅酶A相关的三大物质代谢途径:糖酵解、脂肪酸代谢与氨基酸代谢过程中大部分的酶受到乙酰化或去乙酰化调控。(6)细胞自噬过程中蛋白质修饰以剪切体、核糖体和蛋白酶体等蛋白复合体的形式出现。(7)乙酰化酶KAT7在K155位点、EP300的多个位点如K1203、K1542、K1546、K1707均有显著的乙酰化水平变化,与此对应的底物核内组蛋白H3的K19、K24,H4K16也发生了乙酰化变化。结论:蛋白质的乙酰化修饰也是参与细胞自噬的一种重要的蛋白质翻译后修饰方式,且这些蛋白涉及到细胞内重要的生物过程,即:转录依赖途径和相关物质代谢等转录非依赖途径。细胞自噬与乙酰辅酶A相关代谢途径的乙酰化调控密不可分,从侧面印证了乙酰辅酶A是介导雷帕霉素诱导细胞自噬的一个重要的分子靶标。乙酰化酶KAT7和EP300的自我乙酰化修饰是细胞自噬过程中蛋白质乙酰化修饰的十分重要的方式之一,且这两种酶是参与细胞自噬调控重要的乙酰基转移酶。
[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.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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