利用无细胞体系和iTRAQ鉴定参与亮氨酸调节自噬通路的关键候选蛋白质
发布时间：2018-08-18 09:55
【摘要】：氨基酸不仅是机体合成蛋白质的底物,亦是调节m TORC1信号通路和自噬通路的信号分子(特别是亮氨酸)。氨基酸饥饿会诱导细胞自噬的发生,而氨基酸供给充足则会抑制细胞自噬的发生。近年来,一系列m TORC1上游信号分子得以鉴定并获知其功能,有利于理解氨基酸激活m TORC1和调控自噬的分子机制。因细胞中还可能存在其它未知的蛋白质参与氨基酸调节自噬信号通路。因此,如何有效筛选参与这一过程的未知蛋白质是目前氨基酸调节自噬过程的研究热点之一。本研究通过构建用于研究自噬信号通路的Cell-free system,向该体系中添加单体氨基酸,来验证该种氨基酸是否对自噬具有调节作用,并结合比较蛋白质组学研究方法(同位素标签的相对和绝对定量技术,简称i TRAQ技术)初步鉴定参与该种氨基酸调节自噬信号通路的关键侯选蛋白。首先通过制备自噬体粗提物、S100(胞浆蛋白)、表达纯化Atg14-Flag蛋白,构建了用于研究自噬信号通路的Cell-free system,经试验证明该体系具有较好的有效性。Cell-free system构建成功后,向该体系中添加了单体氨基酸,结果显示在Cell-free system中亮氨酸和蛋氨酸对自噬信号通路可能具有调节作用。进而初步选定亮氨酸为研究对象,运用i TRAQ技术检测添加了亮氨酸后Cell-free system的上清和沉淀,共鉴定到4811个蛋白,其中上清表达差异的蛋白(S2/S1)共243个,沉淀表达差异的蛋白(P2/P1)共134个(本研究中差异蛋白的判定标准为差异倍数≥1.2或≤0.83,且p-value0.05)。上清中表达差异的蛋白最显著富集的代谢通路为氨酰-t RNA生物合成,提示该通路可能与亮氨酸调节自噬相关。沉淀中表达差异的蛋白显著富集的代谢通路之一为氧化磷酸化,提示参与氧化磷酸化代谢通路的相关蛋白也可能与亮氨酸调节自噬相关。在上清中表达下调且沉淀中表达上调的蛋白共有12个(VPS35、CAND1、PKM、PYGL、XRCC6、IPO7、ILF2、GNPDA1、NCDN、NME1、SRP9、SEPHS1),在上清中表达上调且沉淀中表达下调的蛋白数目为零。经过对上述12个蛋白进行生物信息学分析,初步得到VPS35和CAND1很可能是参与亮氨酸调节自噬信号通路的关键蛋白。综上所述,(1)本研究成功的构建了用于研究自噬信号通路的Cell-free system,且该体系具有较好的有效性。(2)初步筛选到亮氨酸和蛋氨酸在Cell-free system中对自噬信号通路可能存在一定的调节作用。(3)经i TRAQ试验和生物信息学分析得到VPS35和CAND1可能是潜在的参与亮氨酸调节自噬信号通路的关键蛋白。
[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，
本文编号：2189114
[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，
本文编号：2189114
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