【摘要】：Birt-Hogg-Dubé(BHD)綜合征是一種遺傳疾病,該疾病是由于FLCN基因缺失引起的。FLCN廣泛存在于生物界,提示該基因可能參與了某些基本的細(xì)胞功能。研究發(fā)現(xiàn),FLCN與氨基酸激活的mTORC1關(guān)系密切,但具體的機(jī)制還存在爭(zhēng)議。本實(shí)驗(yàn)室的前期工作發(fā)現(xiàn)FLCN通過調(diào)節(jié)溶酶體中亮氨酸的水平來調(diào)控mTORC1。本研究旨在深入探索這一過程的分子機(jī)理。PAT1作為一種位于溶酶體表面的氨基酸轉(zhuǎn)運(yùn)蛋白,其功能是從溶酶體內(nèi)向胞質(zhì)運(yùn)輸氨基酸。過表達(dá)PAT1促進(jìn)溶酶體內(nèi)氨基酸的流失,從而抑制mTORC1的活性。本項(xiàng)研究發(fā)現(xiàn),在人源HEK293細(xì)胞中,FLCN通過與PAT1的相互作用來調(diào)控mTORC1。在營(yíng)養(yǎng)缺乏的環(huán)境中,高表達(dá)FLCN使得細(xì)胞仍然可以維持比較高水平的mTORC1。其次,高表達(dá)FLCN,或者提高環(huán)境中的亮氨酸濃度可以拮抗PAT1對(duì)于mTORC1的抑制作用。反之,在營(yíng)養(yǎng)缺乏的環(huán)境中,抑制PAT1導(dǎo)致mTORC1依然保持比較高的水平。而該結(jié)果可以被同時(shí)抑制FLCN所挽救。以上結(jié)果說明,PAT1與FLCN相互拮抗,共同調(diào)節(jié)mTORC1的活性。試驗(yàn)結(jié)果如下:1.我們成功構(gòu)建了穩(wěn)定表達(dá)FLCN-HA蛋白的細(xì)胞系(FLCN-HA-pcDNA3.1)。2.在無氨基酸饑餓條件下,發(fā)現(xiàn)高表達(dá)FLCN促進(jìn)細(xì)胞mTORC1活性,表現(xiàn)為對(duì)外界氨基酸水平,特別是亮氨酸水平的敏感性降低。3.我們成功構(gòu)建了穩(wěn)定表達(dá)EGFP-PAT1蛋白的細(xì)胞系((PAT1-pEGFP-C1)。4.過表達(dá)PAT1抑制mTORC1活性。5.高表達(dá)PAT1抑制過表達(dá)FLCN介導(dǎo)的mTORC1的激活作用。6.FLCN或者高濃度的Leu拮抗PAT1對(duì)mTORC1的抑制作用。7.在無氨基酸饑餓環(huán)境中,抑制PAT1仍然維持mTORC1的活性,但是可以被同時(shí)抑制FLCN所拮抗�？傊�,這些結(jié)果說明,PAT1和FLCN相互拮抗,共同調(diào)控細(xì)胞溶酶體中的氨基酸水平,特別是亮氨酸水平,進(jìn)而影響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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R596

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本文編號(hào)：2170770