BHD综合征致病基因FLCN介导亮氨酸调控mTORC1信号通路的研究

发布时间：2018-03-07 21:29

本文选题：folliculin(FLCN)　切入点：亮氨酸　出处：《西北农林科技大学》2016年博士论文　论文类型：学位论文

【摘要】：BHD(Birt-Hogg-Dubé)综合征是一种由抑癌基因folliculin(FLCN)突变引起的遗传疾病,其临床症状主要包括良性皮肤肿瘤、自发性气胸、肺部囊肿等,严重时某些患者发生肾癌。然而FLCN的生物学功能至今仍未完全阐明。在前期工作中,作者所在实验室构建了一种BHD的果蝇动物模型。利用该模型发现,敲除FLCN基因与mTOR信号通路被抑制的表型相似;更重要的是,在培养基中添加高浓度的亮氨酸(Leu)可以显著挽救FLCN突变的表型。该研究表明,果蝇的FLCN基因与亮氨酸诱导的mTOR信号通路关系密切。然而其具体的分子机理,以及在哺乳动物中是否存在相似的机制还有待研究。为了阐明以上科学问题,本研究以人胚肾细胞系HEK293为模型,深入探究了人FLCN在亮氨酸诱导的mTORC1信号通路中的作用机制,获得了如下主要结果:1.抑制FLCN的表达降低m TORC1活性;添加高浓度的亮氨酸阻止mTORC1活性降低。通过RNA干扰(RNAi)方法抑制FLCN的表达,m TORC1的活性降低;进一步在培养基中添加高浓度的亮氨酸可以显著提高mTORC1活性。2.FLCN能够降低mTORC1对于亮氨酸信号的敏感度。在完全培养基中,添加高浓度的亮氨酸不能使对照细胞的m TORC1活性进一步升高,这说明mTORC1已被完全激活;在FLCN表达被抑制的细胞中,mTORC1的活性随着培养基中亮氨酸水平的升高而不断升高,直至达到在相同培养条件下与对照细胞中相似的水平。3.FLCN定位于溶酶体,其在溶酶体上的富集度受胞内氨基酸浓度影响。免疫荧光以及western blot试验表明,在完全培养基培养条件下,只有少部分FLCN蛋白定位在溶酶体上;在氨基酸缺乏的培养基培养时,有更多的FLCN蛋白定位于溶酶体。4.FLCN维持溶酶体内的亮氨酸水平。现有证据表明,溶酶体内的氨基酸,尤其是亮氨酸,是激活mTORC1的直接信号。当FLCN表达被抑制,或者当环境中氨基酸缺乏时,溶酶体内的亮氨酸浓度显著降低;反之,过表达FLCN使得细胞即使在氨基酸缺乏的情况下,仍然能够维持较高浓度的溶酶体内的亮氨酸水平。5.FLCN能够拮抗跨膜氨基酸转运蛋白PAT1的功能,实现对mTORC1信号的调控。研究表明,氨基酸缺乏能引起mTORC1活性降低,抑制PAT1表达可以部分恢复m TORC1的活性,而同时抑制FLCN表达后,m TORC1活性再次被抑制。6.FLCN抑制PAT1在溶酶体上的分布。在正常培养条件下,有部分PAT1蛋白定位于溶酶体;氨基酸缺乏,或者抑制FLCN的表达能够促进PAT1富集在溶酶体上;而过表达FLCN则会阻止PAT1在溶酶体上的募集。7.FLCN与PAT1存在紧密的相互作用。免疫荧光试验表明,FLCN与PAT1在细胞内有共定位;免疫共沉淀试验进一步表明,FLCN与PAT1存在蛋白与蛋白之间的相互作用。根据以上结果,我们得出以下两个主要结论。一,FLCN通过维持溶酶体内亮氨酸浓度来激活m TORC1;二,FLCN通过抑制PAT1定位于溶酶体实现其对于溶酶体内亮氨酸浓度的调控。本项研究不但发现了FLCN调控溶酶体内氨基酸浓度的一个新功能,同时也揭示出对于溶酶体内亮氨酸信号水平的调控是一种新的mTORC1调控方式。
[Abstract]:BHD (Birt-Hogg-Dub) syndrome is a tumor suppressor gene folliculin (FLCN) genetic disease caused by a mutation, the clinical symptoms mainly include benign skin tumor, spontaneous pneumothorax, pulmonary cyst, severe renal carcinoma in some patients. However, the biological function of FLCN has not been completely elucidated. In previous work, the author a lab flies a BHD animal model. Using this model, found that the knockout phenotype of FLCN gene and mTOR signaling pathway was inhibited by similar; more importantly, in the cultivation of high concentration medium supplemented with leucine (Leu) can significantly save the phenotype of FLCN mutant. The study showed that mTOR signaling pathway FLCN the Drosophila gene and leucine induced closely. However, its molecular mechanism, and whether in mammals have similar mechanism remains to be studied. In order to clarify the above science problem, this study In human embryonic kidney cell line HEK293 as a model, the in-depth study of the mechanism of FLCN in mTORC1 signaling pathway induced by leucine in, obtained the results as follows: 1. the inhibition of expression of FLCN m decreased TORC1 activity; adding high concentrations of leucine blocking mTORC1 activity decreased. By RNA interference (RNAi) suppression of FLCN expression method m, reduce the activity of TORC1; further adding high concentration of leucine in the medium can significantly increase the activity of mTORC1.2.FLCN can reduce the mTORC1 signal for leucine sensitivity. Cultured in complete medium, adding high concentration of leucine can control cells m TORC1 activity increased further, which indicates that mTORC1 has been fully activated the expression of FLCN was inhibited; in cells, mTORC1 activity increased continuously with increasing culture medium and leucine levels, until it reaches under the same culture conditions and control cells. Like the level of.3.FLCN localized in lysosomes, the lysosome enrichment by intracellular amino acid concentrations. Immunofluorescence and Western showed that blot test, in vitro cultured in complete medium, only a small part of FLCN protein is localized in lysosomes; nutrient medium in amino acid deficiency training, FLCN protein localization more in the lysosome.4.FLCN maintain intralysosomal leucine levels. The available evidence suggests that the intralysosomal amino acids, especially leucine, is a direct signal to activate mTORC1. When FLCN expression was inhibited, or when the environment of amino acid deficiency, leucine concentration of lysosomes decreased significantly; conversely, overexpression of FLCN the cells even in the absence of amino acids, can still maintain a high concentration of intralysosomal leucine levels.5.FLCN can antagonize the transmembrane amino acid transporter PAT1 function, the realization of mTOR The regulation of C1 signal. The results show that amino acid deficiency can cause the decrease of mTORC1 activity, inhibit the expression of PAT1 m TORC1 activity can be partially recovered, while inhibiting the expression of FLCN, the distribution of M TORC1 activity was again.6.FLCN suppression on PAT1 in lysosomes. Under normal culture conditions, some of the PAT1 protein localized in lysosomes amino acid; lack or inhibit the expression of FLCN can promote PAT1 accumulation in lysosomes; the interaction between.7.FLCN and PAT1 close to raise and over expression of FLCN will prevent PAT1 in lysosomes. The immunofluorescence experiments indicate that FLCN colocalized with PAT1 in cells; CO immunoprecipitation experiments further indicate that the interaction between. Between protein and protein FLCN and PAT1. According to the above results, we draw the following two conclusions. First, the FLCN solution by maintaining the leucine concentration in vivo enzyme to activate the m TORC1; two, FLCN After inhibition of PAT1 localized in lysosomes to achieve its regulation of soluble leucine concentration in vivo enzyme. This study not only found a new function of FLCN solution concentration of amino acids in the regulation of the enzyme, but also reveals the regulation of lysosomal leucine signal level is a new kind of mTORC1 control mode.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R596

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