本文選題：mTORC1　切入點：無細胞體系　出處：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：哺乳動物雷帕霉素靶蛋白復(fù)合物1(mTORC1,mammalian target of rapamycin complex 1)的激活不僅能促進蛋白質(zhì)的合成,還能抑制自噬等蛋白質(zhì)的降解途徑的發(fā)生。氨基酸尤其是亮氨酸不僅是蛋白質(zhì)合成的原料,也能作為信號分子參與mTORC1的激活過程。近年來,越來越多的蛋白被發(fā)現(xiàn)參與氨基酸激活mTORC1的過程。然而,關(guān)于氨基酸在細胞內(nèi)的感應(yīng)機制尚不明確,細胞中還可能存在其它未知的蛋白質(zhì)分子參與氨基酸激活mTORC1的過程。本研究通過構(gòu)建用于研究mTORC1信號通路的無細胞體系(Cell-free system),結(jié)合同位素標(biāo)記相對和絕對定量iTRAQ(isobaric tags for relative and absolute quantification)技術(shù)及生物信息學(xué)分析,初步篩選參與亮氨酸調(diào)節(jié)mTORC1信號通路的關(guān)鍵差異候選蛋白質(zhì)。本研究的無細胞體系由溶酶體粗提物(包括P20(+)和P20(-))、胞漿蛋白(包括S100(+)和S100(-))、純化的標(biāo)簽蛋白HA-raptor(mTORC1的組分之一)組成。無細胞體系結(jié)果表明,在由胞漿蛋白S100(-)和溶酶體粗提物P20(-)組成的無細胞體系中,亮氨酸能促進Raptor與溶酶體P20(-)的結(jié)合。無細胞體系反應(yīng)完成后,我們將對照組的上清和沉淀(S1,P1)與亮氨酸組的上清和沉淀(S2,P2)進行iTRAQ試驗。本次iTRAQ試驗共鑒定到6292個蛋白,以變化倍數(shù)(Fold change)1.2或0.83和Q值(Q-value)0.05兩個條件進行差異蛋白的篩選。在P2-VS-P1組中,上調(diào)差異蛋白數(shù)(Up-regulated proteins)為208個,下調(diào)差異蛋白數(shù)(Down-regulated proteins)為190個;在S2-VS-S1組中,上調(diào)差異蛋白數(shù)(Up-regulated proteins)為12個,下調(diào)差異蛋白數(shù)(Down-regulated proteins)為3個。對上述差異蛋白進行生物信息學(xué)分析(包括GO、COG注釋、Pathway注釋分析)后發(fā)現(xiàn),差異蛋白主要為細胞器上參與代謝過程的蛋白,且參與脂質(zhì)代謝和與神經(jīng)退行性疾病相關(guān)的蛋白也可能參與亮氨酸對mTORC1的激活過程。參與亮氨酸激活mTORC1信號通路的關(guān)鍵差異候選蛋白需要綜合考慮上清和沉淀中的差異蛋白。其中,在上清中下調(diào)且在沉淀中上調(diào)的蛋白數(shù)為0,而在上清中上調(diào)且在沉淀中下調(diào)的蛋白有6個,因而我們初步將這6個蛋白(APOA1、CFH、FGA、ERH、CANX、F2)定義為參與亮氨酸激活m TORC1信號通路的關(guān)鍵差異候選蛋白。對CANX進行初步驗證后發(fā)現(xiàn),無細胞體系樣品中該蛋白的分布規(guī)律與iTRAQ數(shù)據(jù)一致,且CANX與溶酶體上的標(biāo)志性蛋白Lamp2的共定位結(jié)果也說明亮氨酸能促進CANX向細胞質(zhì)中遷移。綜上所述,(1)本研究首次成功構(gòu)建了用于研究亮氨酸調(diào)節(jié)mTORC1信號通路的無細胞體系,且該體系有效性好,穩(wěn)定性高。(2)iTRAQ數(shù)據(jù)與生物信息學(xué)分析結(jié)果表明,APOA1、CFH、FGA、ERH、CANX、F2可能參與亮氨酸對于mTORC1信號通路的調(diào)節(jié)。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.2

【參考文獻】

相關(guān)期刊論文 前1條

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本文編號：1686689