利用无细胞体系筛选参与亮氨酸调节蛋白质合成信号通路的关键候选蛋白质

发布时间：2018-03-30 16:24

本文选题：mTORC1　切入点：无细胞体系　出处：《华中农业大学》2017年硕士论文

【摘要】：哺乳动物雷帕霉素靶蛋白复合物1(mTORC1,mammalian target of rapamycin complex 1)的激活不仅能促进蛋白质的合成,还能抑制自噬等蛋白质的降解途径的发生。氨基酸尤其是亮氨酸不仅是蛋白质合成的原料,也能作为信号分子参与mTORC1的激活过程。近年来,越来越多的蛋白被发现参与氨基酸激活mTORC1的过程。然而,关于氨基酸在细胞内的感应机制尚不明确,细胞中还可能存在其它未知的蛋白质分子参与氨基酸激活mTORC1的过程。本研究通过构建用于研究mTORC1信号通路的无细胞体系(Cell-free system),结合同位素标记相对和绝对定量iTRAQ(isobaric tags for relative and absolute quantification)技术及生物信息学分析,初步筛选参与亮氨酸调节mTORC1信号通路的关键差异候选蛋白质。本研究的无细胞体系由溶酶体粗提物(包括P20(+)和P20(-))、胞浆蛋白(包括S100(+)和S100(-))、纯化的标签蛋白HA-raptor(mTORC1的组分之一)组成。无细胞体系结果表明,在由胞浆蛋白S100(-)和溶酶体粗提物P20(-)组成的无细胞体系中,亮氨酸能促进Raptor与溶酶体P20(-)的结合。无细胞体系反应完成后,我们将对照组的上清和沉淀(S1,P1)与亮氨酸组的上清和沉淀(S2,P2)进行iTRAQ试验。本次iTRAQ试验共鉴定到6292个蛋白,以变化倍数(Fold change)1.2或0.83和Q值(Q-value)0.05两个条件进行差异蛋白的筛选。在P2-VS-P1组中,上调差异蛋白数(Up-regulated proteins)为208个,下调差异蛋白数(Down-regulated proteins)为190个;在S2-VS-S1组中,上调差异蛋白数(Up-regulated proteins)为12个,下调差异蛋白数(Down-regulated proteins)为3个。对上述差异蛋白进行生物信息学分析(包括GO、COG注释、Pathway注释分析)后发现,差异蛋白主要为细胞器上参与代谢过程的蛋白,且参与脂质代谢和与神经退行性疾病相关的蛋白也可能参与亮氨酸对mTORC1的激活过程。参与亮氨酸激活mTORC1信号通路的关键差异候选蛋白需要综合考虑上清和沉淀中的差异蛋白。其中,在上清中下调且在沉淀中上调的蛋白数为0,而在上清中上调且在沉淀中下调的蛋白有6个,因而我们初步将这6个蛋白(APOA1、CFH、FGA、ERH、CANX、F2)定义为参与亮氨酸激活m TORC1信号通路的关键差异候选蛋白。对CANX进行初步验证后发现,无细胞体系样品中该蛋白的分布规律与iTRAQ数据一致,且CANX与溶酶体上的标志性蛋白Lamp2的共定位结果也说明亮氨酸能促进CANX向细胞质中迁移。综上所述,(1)本研究首次成功构建了用于研究亮氨酸调节mTORC1信号通路的无细胞体系,且该体系有效性好,稳定性高。(2)iTRAQ数据与生物信息学分析结果表明,APOA1、CFH、FGA、ERH、CANX、F2可能参与亮氨酸对于mTORC1信号通路的调节。
[Abstract]:The activation of mammalian rapamycin target protein complex 1 mTORC1mammalian target of rapamycin complex 1 not only promotes protein synthesis, but also inhibits the process of protein degradation such as autophagy.Amino acids, especially leucine, are not only the raw materials for protein synthesis, but also participate in the activation of mTORC1 as signaling molecules.In recent years, more and more proteins have been found to be involved in the process of amino acid activation of mTORC1.In this study, a cell-free system for the study of mTORC1 signaling pathway was constructed, combined with relative and absolute quantitative iTRAQ(isobaric tags for relative and absolute quantification techniques and bioinformatics analysis.Primary screening of key differential candidate proteins involved in leucine regulation of mTORC1 signaling pathway.The cell-free system was composed of lysosomal crude extracts (including P20 () and P20P20 ()), cytoplasmic proteins (including S100 () and S100 ()), and components of purified label protein (HA-raptor(mTORC1).The results of cell-free system showed that leucine could promote the binding of Raptor to lysosomal P20-) in an acellular system composed of cytoplasmic protein S100-) and lysosomal crude extract P20-).After the acellular system reaction was completed, the supernatants and precipitates of the control group and the leucine group were tested for iTRAQ.A total of 6292 proteins were identified in this iTRAQ test. The differential proteins were screened under the conditions of fold change)1.2 or 0.83 and Q-value0. 05.In the P2-VS-P1 group, the Up-regulated proteinss were 208 and the down-regulated proteins Down-regulated proteinss were 190. In the S2-VS-S1 group, the Up-regulated proteinss were 12 and the down-regulated proteins were 3.Bioinformatics analysis of the above differentially expressed proteins (including GOCOG annotation and Pathway annotation) showed that the differential proteins were mainly involved in metabolic processes in the organelles.The proteins involved in lipid metabolism and neurodegenerative diseases may also be involved in the activation of leucine to mTORC1.The key differential candidate proteins involved in leucine activation of mTORC1 signaling pathway need to consider the differential proteins in supernatants and precipitates.Among them, the number of proteins down-regulated in supernatant and up-regulated in precipitation was 0, while in supernatant and down-regulated in precipitation there were 6 proteins.Therefore, we preliminarily defined the six proteins APOA1CFH, FGAAERH, CANXF2) as the key differential candidate proteins involved in leucine-activated m TORC1 signaling pathway.It was found that the distribution of the protein in the cell-free system was consistent with that of iTRAQ, and that the co-localization of CANX and Lamp2, the iconic protein on lysosome, also indicated that leucine could promote the migration of CANX into the cytoplasm.In this study, we successfully constructed a cell-free system for the study of leucine regulating mTORC1 signaling pathway, and the system was effective.The results of high stability data and bioinformatics analysis suggested that APOA1 / FGAA / CANXF2 might be involved in the regulation of mTORC1 signaling pathway by leucine.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.2

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