FLCN与PAT1相互作用调控mTORC1信号通路的机理研究
发布时间：2018-08-07 17:15
【摘要】：Birt-Hogg-Dubé(BHD)综合征是一种遗传疾病,该疾病是由于FLCN基因缺失引起的。FLCN广泛存在于生物界,提示该基因可能参与了某些基本的细胞功能。研究发现,FLCN与氨基酸激活的mTORC1关系密切,但具体的机制还存在争议。本实验室的前期工作发现FLCN通过调节溶酶体中亮氨酸的水平来调控mTORC1。本研究旨在深入探索这一过程的分子机理。PAT1作为一种位于溶酶体表面的氨基酸转运蛋白,其功能是从溶酶体内向胞质运输氨基酸。过表达PAT1促进溶酶体内氨基酸的流失,从而抑制mTORC1的活性。本项研究发现,在人源HEK293细胞中,FLCN通过与PAT1的相互作用来调控mTORC1。在营养缺乏的环境中,高表达FLCN使得细胞仍然可以维持比较高水平的mTORC1。其次,高表达FLCN,或者提高环境中的亮氨酸浓度可以拮抗PAT1对于mTORC1的抑制作用。反之,在营养缺乏的环境中,抑制PAT1导致mTORC1依然保持比较高的水平。而该结果可以被同时抑制FLCN所挽救。以上结果说明,PAT1与FLCN相互拮抗,共同调节mTORC1的活性。试验结果如下:1.我们成功构建了稳定表达FLCN-HA蛋白的细胞系(FLCN-HA-pcDNA3.1)。2.在无氨基酸饥饿条件下,发现高表达FLCN促进细胞mTORC1活性,表现为对外界氨基酸水平,特别是亮氨酸水平的敏感性降低。3.我们成功构建了稳定表达EGFP-PAT1蛋白的细胞系((PAT1-pEGFP-C1)。4.过表达PAT1抑制mTORC1活性。5.高表达PAT1抑制过表达FLCN介导的mTORC1的激活作用。6.FLCN或者高浓度的Leu拮抗PAT1对mTORC1的抑制作用。7.在无氨基酸饥饿环境中,抑制PAT1仍然维持mTORC1的活性,但是可以被同时抑制FLCN所拮抗。总之,这些结果说明,PAT1和FLCN相互拮抗,共同调控细胞溶酶体中的氨基酸水平,特别是亮氨酸水平,进而影响mTORC1。
[Abstract]:Birt-Hogg-Dub 茅 (BHD) syndrome is a genetic disease, which is caused by the deletion of FLCN gene, which is widely present in the biological world, suggesting that the gene may be involved in some basic cellular functions. It was found that FLCN was closely related to amino acid activated mTORC1, but the specific mechanism was still controversial. Our previous work found that FLCN regulates mTORC1 by regulating leucine levels in lysosomes. The purpose of this study is to explore the molecular mechanism of this process. PAT1 is a amino acid transporter located on the surface of lysosome and its function is to transport amino acids from lysosome to cytoplasm. Overexpression of PAT1 promotes the loss of amino acids in lysosomes and thus inhibits the activity of mTORC1. In this study, we found that in human HEK293 cells, HEK293 regulates mTORC1 by interaction with PAT1. In nutritionally deficient environments, high expression of FLCN allows cells to maintain high levels of mTORC1. Secondly, high expression of FLCNs or increased concentration of leucine in the environment could antagonize the inhibitory effect of PAT1 on mTORC1. In contrast, inhibition of PAT1 in nutrient-deficient environments resulted in higher levels of mTORC1. This result can be saved by simultaneous inhibition of FLCN. These results suggest that PAT1 and FLCN antagonize each other and regulate the activity of mTORC1. The test results are as follows: 1. We successfully constructed a cell line (FLCN-HA-pcDNA3.1. 2. 2) stably expressing FLCN-HA protein. Under the condition of no amino acid starvation, it was found that the overexpression of FLCN promoted the mTORC1 activity of the cells, which showed that the sensitivity to the external amino acid level, especially the leucine level, decreased. 3. We successfully constructed a cell line (PAT1-pEGFP-C1). 4. 4) expressing EGFP-PAT1 protein stably. Overexpression of PAT1 inhibits the activity of mTORC1. Overexpression of PAT1 inhibits the activation of mTORC1 mediated by overexpression of FLCN. 6. FLCN or high concentration of Leu antagonizes the inhibitory effect of PAT1 on mTORC1. In the absence of amino acid starvation, the inhibition of PAT1 still maintained the activity of mTORC1, but could be antagonized by the inhibition of FLCN at the same time. In conclusion, these results suggest that PAT1 and FLCN antagonize each other to regulate the levels of amino acids in lysosomes, especially leucine, and thus affect mTORC1.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R596
本文编号：2170770
[Abstract]:Birt-Hogg-Dub 茅 (BHD) syndrome is a genetic disease, which is caused by the deletion of FLCN gene, which is widely present in the biological world, suggesting that the gene may be involved in some basic cellular functions. It was found that FLCN was closely related to amino acid activated mTORC1, but the specific mechanism was still controversial. Our previous work found that FLCN regulates mTORC1 by regulating leucine levels in lysosomes. The purpose of this study is to explore the molecular mechanism of this process. PAT1 is a amino acid transporter located on the surface of lysosome and its function is to transport amino acids from lysosome to cytoplasm. Overexpression of PAT1 promotes the loss of amino acids in lysosomes and thus inhibits the activity of mTORC1. In this study, we found that in human HEK293 cells, HEK293 regulates mTORC1 by interaction with PAT1. In nutritionally deficient environments, high expression of FLCN allows cells to maintain high levels of mTORC1. Secondly, high expression of FLCNs or increased concentration of leucine in the environment could antagonize the inhibitory effect of PAT1 on mTORC1. In contrast, inhibition of PAT1 in nutrient-deficient environments resulted in higher levels of mTORC1. This result can be saved by simultaneous inhibition of FLCN. These results suggest that PAT1 and FLCN antagonize each other and regulate the activity of mTORC1. The test results are as follows: 1. We successfully constructed a cell line (FLCN-HA-pcDNA3.1. 2. 2) stably expressing FLCN-HA protein. Under the condition of no amino acid starvation, it was found that the overexpression of FLCN promoted the mTORC1 activity of the cells, which showed that the sensitivity to the external amino acid level, especially the leucine level, decreased. 3. We successfully constructed a cell line (PAT1-pEGFP-C1). 4. 4) expressing EGFP-PAT1 protein stably. Overexpression of PAT1 inhibits the activity of mTORC1. Overexpression of PAT1 inhibits the activation of mTORC1 mediated by overexpression of FLCN. 6. FLCN or high concentration of Leu antagonizes the inhibitory effect of PAT1 on mTORC1. In the absence of amino acid starvation, the inhibition of PAT1 still maintained the activity of mTORC1, but could be antagonized by the inhibition of FLCN at the same time. In conclusion, these results suggest that PAT1 and FLCN antagonize each other to regulate the levels of amino acids in lysosomes, especially leucine, and thus affect mTORC1.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R596
【参考文献】
相关硕士学位论文 前1条
1 陈风雷;小鼠Zhangfei基因过表达和shRNA干扰慢病毒载体的构建和鉴定[D];西北农林科技大学;2013年
，本文编号：2170770
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