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

【摘要】：目的:探究雷帕霉素誘導(dǎo)細(xì)胞自噬過程中蛋白質(zhì)乙酰化組學(xué)的特征。方法:將細(xì)胞分為陰性對(duì)照組、DMSO對(duì)照組、雷帕霉素處理組,采用western blot檢測(cè)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(diǎn),確立細(xì)胞自噬的過程;設(shè)立對(duì)照組與0.1μM雷帕霉素處理,采用SILAC標(biāo)記技術(shù),經(jīng)過胰酶消化、乙�；嚢彼峥贵w富集,并行串聯(lián)質(zhì)譜定量檢測(cè)細(xì)胞內(nèi)乙酰化蛋白水平,而后通過GO、Motif-x、KEGG、CORUM等數(shù)據(jù)庫(kù)對(duì)乙�；鞍走M(jìn)行分析。結(jié)果:(1)雷帕霉素處理細(xì)胞后,mTOR及其底物RPS6KB1、EIF4G1的磷酸化顯著下調(diào),同時(shí)LC3熒光點(diǎn)及LC3-II/LC3-I比值明顯升高,驗(yàn)證了雷帕霉素可以誘導(dǎo)細(xì)胞的自噬。(2)SILAC技術(shù)和質(zhì)譜分析結(jié)果顯示,檢測(cè)到944個(gè)蛋白上共1808個(gè)乙�；稽c(diǎn),其中397個(gè)蛋白上共533個(gè)位點(diǎn)乙�；l(fā)生顯著變化。鑒定出來蛋白中,60.4%僅僅具有一個(gè)乙�；稽c(diǎn),20.4%具有兩個(gè)位點(diǎn),19.2%的蛋白具有多個(gè)位點(diǎn)發(fā)生乙�；＿@些乙�；鞍自诩�(xì)胞質(zhì)、細(xì)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(gè)潛在的自噬相關(guān)乙�；颉�(4)GO富集分析的結(jié)果顯示,大分子復(fù)合體的組裝、細(xì)胞代謝過程、基因表達(dá)、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�；蛉ヒ阴；{(diào)控。(6)細(xì)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(diǎn)、EP300的多個(gè)位點(diǎn)如K1203、K1542、K1546、K1707均有顯著的乙�；阶兓�,與此對(duì)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�；揎椧彩菂⑴c細(xì)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(xì)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(xì)胞自噬與乙酰輔酶A相關(guān)代謝途徑的乙�；{(diào)控密不可分,從側(cè)面印證了乙酰輔酶A是介導(dǎo)雷帕霉素誘導(dǎo)細(xì)胞自噬的一個(gè)重要的分子靶標(biāo)。乙�；窴AT7和EP300的自我乙酰化修飾是細(xì)胞自噬過程中蛋白質(zhì)乙�；揎椀氖种匾姆绞街�,且這兩種酶是參與細(xì)胞自噬調(diào)控重要的乙酰基轉(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(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R96

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