【摘要】：氨基酸不僅是機(jī)體合成蛋白質(zhì)的底物,亦是調(diào)節(jié)m TORC1信號通路和自噬通路的信號分子(特別是亮氨酸)。氨基酸饑餓會誘導(dǎo)細(xì)胞自噬的發(fā)生,而氨基酸供給充足則會抑制細(xì)胞自噬的發(fā)生。近年來,一系列m TORC1上游信號分子得以鑒定并獲知其功能,有利于理解氨基酸激活m TORC1和調(diào)控自噬的分子機(jī)制。因細(xì)胞中還可能存在其它未知的蛋白質(zhì)參與氨基酸調(diào)節(jié)自噬信號通路。因此,如何有效篩選參與這一過程的未知蛋白質(zhì)是目前氨基酸調(diào)節(jié)自噬過程的研究熱點之一。本研究通過構(gòu)建用于研究自噬信號通路的Cell-free system,向該體系中添加單體氨基酸,來驗證該種氨基酸是否對自噬具有調(diào)節(jié)作用,并結(jié)合比較蛋白質(zhì)組學(xué)研究方法(同位素標(biāo)簽的相對和絕對定量技術(shù),簡稱i TRAQ技術(shù))初步鑒定參與該種氨基酸調(diào)節(jié)自噬信號通路的關(guān)鍵侯選蛋白。首先通過制備自噬體粗提物、S100(胞漿蛋白)、表達(dá)純化Atg14-Flag蛋白,構(gòu)建了用于研究自噬信號通路的Cell-free system,經(jīng)試驗證明該體系具有較好的有效性。Cell-free system構(gòu)建成功后,向該體系中添加了單體氨基酸,結(jié)果顯示在Cell-free system中亮氨酸和蛋氨酸對自噬信號通路可能具有調(diào)節(jié)作用。進(jìn)而初步選定亮氨酸為研究對象,運用i TRAQ技術(shù)檢測添加了亮氨酸后Cell-free system的上清和沉淀,共鑒定到4811個蛋白,其中上清表達(dá)差異的蛋白(S2/S1)共243個,沉淀表達(dá)差異的蛋白(P2/P1)共134個(本研究中差異蛋白的判定標(biāo)準(zhǔn)為差異倍數(shù)≥1.2或≤0.83,且p-value0.05)。上清中表達(dá)差異的蛋白最顯著富集的代謝通路為氨酰-t RNA生物合成,提示該通路可能與亮氨酸調(diào)節(jié)自噬相關(guān)。沉淀中表達(dá)差異的蛋白顯著富集的代謝通路之一為氧化磷酸化,提示參與氧化磷酸化代謝通路的相關(guān)蛋白也可能與亮氨酸調(diào)節(jié)自噬相關(guān)。在上清中表達(dá)下調(diào)且沉淀中表達(dá)上調(diào)的蛋白共有12個(VPS35、CAND1、PKM、PYGL、XRCC6、IPO7、ILF2、GNPDA1、NCDN、NME1、SRP9、SEPHS1),在上清中表達(dá)上調(diào)且沉淀中表達(dá)下調(diào)的蛋白數(shù)目為零。經(jīng)過對上述12個蛋白進(jìn)行生物信息學(xué)分析,初步得到VPS35和CAND1很可能是參與亮氨酸調(diào)節(jié)自噬信號通路的關(guān)鍵蛋白。綜上所述,(1)本研究成功的構(gòu)建了用于研究自噬信號通路的Cell-free system,且該體系具有較好的有效性。(2)初步篩選到亮氨酸和蛋氨酸在Cell-free system中對自噬信號通路可能存在一定的調(diào)節(jié)作用。(3)經(jīng)i TRAQ試驗和生物信息學(xué)分析得到VPS35和CAND1可能是潛在的參與亮氨酸調(diào)節(jié)自噬信號通路的關(guān)鍵蛋白。
[Abstract]:Amino acids are not only the substrates of protein synthesis, but also signaling molecules (especially leucine) that regulate m TORC1 signaling pathway and autophagy pathway. Amino acid starvation induces autophagy, while adequate supply of amino acids inhibits autophagy. In recent years, a series of upstream signal molecules of m TORC1 have been identified and their functions have been known, which is helpful to understand the molecular mechanism of amino acid activation of m TORC1 and regulation of autophagy. Other unknown proteins may be involved in the regulation of autophagy signaling pathway by amino acids. Therefore, how to effectively screen unknown proteins involved in this process is one of the hot topics in the process of amino acid regulation autophagy. In this study, we constructed a Cell-free system for the study of autophagy signaling pathway and added monomeric amino acids to the system to verify whether the amino acid has a regulatory effect on autophagy. The key candidate proteins involved in the regulation of autophagy signal pathway by amino acids were identified by comparative proteomics (relative and absolute quantitative techniques of isotopic labeling, or I TRAQ technique). Firstly, the Atg14-Flag protein was expressed and purified by the preparation of S100 (cytosolic protein), and the Cell-free system for the study of autophagy signaling pathway was constructed. The system was proved to be effective. Cell-free system was successfully constructed. Monomeric amino acids were added to the system. The results showed that leucine and methionine might regulate the autophagy signaling pathway in Cell-free system. The supernatant and precipitate of Cell-free system after adding leucine were detected by I TRAQ technique. A total of 4811 proteins were identified, of which 243 were differentially expressed in supernatant (S2/S1). There were 134 differentially expressed proteins (P2/P1) in this study (the criteria of differential proteins in this study were multiple of difference 鈮，

本文編號：2189114