鈣離子參與下的相關(guān)通路在HIV-1 Vpu拮抗Tetherin過程中的作用
發(fā)布時間：2018-11-05 12:10
【摘要】：Tetherin(BST-2,CD317)是一種具有特殊拓撲結(jié)構(gòu)的II型膜蛋白,它的結(jié)構(gòu)包括N端的胞質(zhì)尾端,單次穿膜區(qū),胞外的螺旋區(qū)以及C端的磷脂酰肌醇錨定區(qū)(潛在的二次穿膜區(qū))。目前發(fā)現(xiàn)Tetherin能夠嵌入宿主細胞質(zhì)膜和新生的病毒膜,以抑制病毒的釋放。HIV-1附屬蛋白Vpu是一種編碼81個氨基酸的I型膜蛋白,早期的研究表明,Vpu在HIV-1傳播過程中有兩個主要的功能,其一是Vpu可以下調(diào)細胞表面的CD4受體,阻止病毒感染細胞中g(shù)p160/CD4復(fù)合體形成,從而使宿主細胞被不同病毒多重感染;另外Vpu可以解除掉Tetherin(BST-2,CD317)系留病毒的作用,促進子代病毒的釋放。Vpu拮抗Tetherin作用的分子機制目前仍未完全明確。起初,有報道猜測Vpu拮抗Tetherin與降解CD4受體的機制相似,即通過穿膜螺旋區(qū)與Tetherin相互結(jié)合,通過泛素化酶和蛋白酶體途徑對Tetherin本身起到降解作用,使之不能夠完成病毒的物理系留作用。然而后來的研究證實Vpu在不降解,也不下調(diào)Tetherin在細胞表面表達量的前提下也能夠促進病毒釋放,說明上述理論并不完備。也有報道間接證明Tetherin能夠特異性識別病毒出芽位點,因而猜測Vpu拮抗Tetherin可能通過破壞其識別作用達到促進病毒釋放的作用。還有一些其他的報道顯示在有Vpu存在的情況下,Tethern仍然可以被包裝進子代病毒但卻無法發(fā)揮系留作用,綜上說明Vpu拮抗Tetherin的關(guān)鍵分子機制仍有待進一步闡明。Vpu自身的同源寡聚體能形成弱選擇性的鉀離子通道,同時Vpu也能夠干擾TASK導(dǎo)致宿主細胞膜的去極化,我們猜測這種去極化現(xiàn)象能夠進而觸發(fā)電壓依賴型鈣離子通道,引起細胞內(nèi)的鈣離子濃度上升,在之前的研究中已經(jīng)初步證實了這種現(xiàn)象的發(fā)生。本篇論文就是在此基礎(chǔ)上,進一步確認Vpu引起細胞內(nèi)鈣離子濃度上升是由于觸發(fā)哪種電壓依賴型鈣離子通道導(dǎo)致,這種作用與其自身離子通道活性的相關(guān)性,以及鈣離子參與下的相關(guān)下游通路在HIV-1 Vpu拮抗Tetherin過程中的作用,從而為Vpu拮抗Tetherin提供一個新思路。在本論文中,我們首先在穩(wěn)定表達Vpu的293T細胞中探究Vpu對細胞內(nèi)鈣離子濃度的影響以及與電壓依賴型鈣離子通道的關(guān)系。我們在穩(wěn)定表達Vpu的細胞中利用綠色熒光鈣指示劑Fluo-4 AM標(biāo)記細胞內(nèi)的鈣離子,利用倒置顯微鏡、分光光度儀以及流式細胞儀檢測細胞內(nèi)鈣離子的濃度變化,檢測到Vpu對細胞內(nèi)的鈣離子有上調(diào)作用;同時我們又通過VDCC拮抗劑以及RNA沉默VDCC的方法檢測細胞內(nèi)的鈣離子濃度變化,來確定鈣濃度變化的確是通過介導(dǎo)電壓依賴型鈣離子通道。其次,在本篇論文里,我們又進一步研究了鈣離子作為媒介的下游通路對Vpu拮抗Tetherin的影響。我們利用了透射電子顯微鏡觀察Tetherin是否通過其特殊的拓撲結(jié)構(gòu)識別子代病毒出芽造成的細胞膜膜曲率變化,以及Vpu對這一過程的干擾和鈣離子對其的影響;同時,我們還利用了Co-IP驗證是否Tetherin與Gag物理相互結(jié)合,以及Vpu是否對此過程有影響,結(jié)果表明Vpu不干擾Tetherin定位于出芽位點的過程。除此之外,我們還嘗試探究出Vpu可能通過提升細胞內(nèi)鈣離子濃度,抑制Tetherin的Hem ITAM磷酸化,進而影響其NF-κB信號傳導(dǎo)活性,以及一系列通路中鈣離子通道起到作用。最后,我們又對Vpu通過激活水解酶從而切割Tetherin的這種可能的機制進行了進一步研究。從基因水平和蛋白水平驗證鈣離子在Tetherin影響基質(zhì)金屬酶和Furin酶活性上的作用以及Tetherin潛在的Furin切割位點突變后對病毒釋放的影響,結(jié)果表明Furin并不切割Tetherin,但有趣的是Tetherin卻可能抑制Furin自身的活性。通過作用位點軟件預(yù)測,我們發(fā)現(xiàn)信號肽酶對于Tetherin也可能有潛在切割作用,所以我們對此也做了初步探究。通過上述實驗,我們更精細的探究了Vpu對于細胞內(nèi)的鈣離子濃度的影響通過何種方式介導(dǎo),以及初步明確鈣離子通道與拮抗Tetherin活性相關(guān)的下游分子機制,為Vpu拮抗Tetherin的機制的進一步闡明提供了嶄新的方向。在未來,我們會對以上初步探究的各項通路綜合起來,更全面的揭示出Vpu依賴鈣離子拮抗Tetherin的途徑。
[Abstract]:Terethin (BST-2, CD317) is a type II membrane protein with a special topological structure. Its structure includes the cytoplasmic tail end of N terminal, single pass membrane region, spiral region outside the cell, and phospholipid and inositol anchoring region of C terminal (potential secondary puncture zone). Tethin is now found to be able to embed the host cytoplasmic membrane and the nascent viral membrane to inhibit the release of the virus. HIV-1 accessory protein Vpu is an I-type membrane protein encoding 81 amino acids. Early studies have shown that Vpu has two major functions in the transmission of HIV-1, one of which is that Vpu can down-regulate the CD4 receptor on the cell surface and prevent the formation of gp160/ CD4 complex in virus infected cells. so that the host cell is infected with different viruses; and the Vpu can release the effect of the teethin (BST-2, CD317) captive virus, and promote the release of the progeny virus. The molecular mechanism of Vpu antagonizing the role of Tethin is still not completely clear at present. At first, it was reported that Vpu antagonized the mechanism of Tethin and the degradation of CD4 receptor, that is, through the combination of membrane helix region and Tethin, Tethin itself was degraded by ubiquitination enzyme and proteome pathway, so that it could not complete the physical system retention of the virus. However, subsequent studies confirmed that Vpu could also promote viral release without degrading, or reducing the amount of Tethin's expression on the surface of cells, suggesting that the theory was not complete. It was also reported that Tethin was able to specifically identify the viral replicon site, thus guessing that Vpu antagonized the role of Tethin to promote viral release by disrupting its recognition. There are some other reports showing that, in the presence of Vpu, Tethn can still be packaged into the progeny virus, but it is unable to play a mooring function, and the key molecular mechanism of Vpu antagonizing Tethin remains to be further elucidated. Vpu's own homologous oligomer forms a weakly selective potassium ion channel while Vpu can also interfere with TASK leading to depolarization of the host cell membrane, and we suspect that this depolarization phenomenon can trigger a voltage-dependent calcium ion channel, causing a rise in calcium ion concentration in the cell, This phenomenon has been preliminarily confirmed in previous studies. On the basis of this, we further confirm that Vpu causes intracellular calcium ion concentration to rise as a result of which voltage-dependent calcium ion channels trigger the correlation between this effect and its own ion channel activity. and the action of the relevant downstream pathway under the participation of calcium ions in the process of antagonizing Tethin by HIV-1Vpu, thereby providing a new idea for Vpu to antagonize Tethin. In this paper, we first explored the influence of Vpu on intracellular calcium ion concentration and the relationship with voltage-dependent calcium ion channels in 293T cells stably expressing Vpu. The calcium ions in cells were labeled with green fluorescent calcium indicator Fluo-4AM in the cells stably expressing Vpu, and the concentration of calcium ions in cells was detected by an inverted microscope, a spectrophotometer and a flow cytometer, and the up-regulation effect of Vpu on calcium ions in the cells was detected. At the same time, we detected the change of calcium ion concentration in the cells by means of VDCC antagonist and RNA silencing VDCC to determine that the change of calcium concentration is indeed by mediating the voltage-dependent calcium ion channel. Secondly, in this paper, we further study the influence of calcium ion as the downstream pathway of the medium on Vpu antagonist Tethin. We use the transmission electron microscope to observe whether Tethin has identified the change of membrane curvature of membrane membrane caused by sub-generation of virus by its special topological structure, and the interference of Vpu on the process and the effect of calcium ion on it; meanwhile, We also use Co-IP to verify whether Tethin and Gag are physically bonded to each other, and whether Vpu is affected by this process, and the results show that Vpu does not interfere with the process of Tethin to be located at the site. In addition, we tried to show that Vpu could inhibit the phosphorylation of Hem ITAM by increasing the intracellular calcium ion concentration, thus affecting the signal transduction activity of NF-Sepharose B and the role of calcium ion channels in a series of pathways. Finally, we further studied the possible mechanism of Vpu by activating the hydrolase to cut Tethin. The effect of calcium ions on the activity of matrix metalloenzymes and Furin enzymes and the effects of Tethin potential Furin cleavage site mutations on viral release were verified from gene levels and protein levels, and the results indicated that Furin did not cut Tethin, but it was interesting that Tethin could inhibit Furin's own activity. We have also made a preliminary inquiry about the potential cleavage of Tethin through the prediction of active site software. Through the above experiments, we examine the influence of Vpu on the concentration of calcium ions in the cells and the mechanism of downstream molecular mechanism related to the activity of Tethin, and provide a new direction for the further elucidation of the mechanism of Vpu antagonizing Tethin. In the future, we can comprehensively reveal the ways of Vpu-dependent calcium ion antagonizing Tethin.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R512.91
[Abstract]:Terethin (BST-2, CD317) is a type II membrane protein with a special topological structure. Its structure includes the cytoplasmic tail end of N terminal, single pass membrane region, spiral region outside the cell, and phospholipid and inositol anchoring region of C terminal (potential secondary puncture zone). Tethin is now found to be able to embed the host cytoplasmic membrane and the nascent viral membrane to inhibit the release of the virus. HIV-1 accessory protein Vpu is an I-type membrane protein encoding 81 amino acids. Early studies have shown that Vpu has two major functions in the transmission of HIV-1, one of which is that Vpu can down-regulate the CD4 receptor on the cell surface and prevent the formation of gp160/ CD4 complex in virus infected cells. so that the host cell is infected with different viruses; and the Vpu can release the effect of the teethin (BST-2, CD317) captive virus, and promote the release of the progeny virus. The molecular mechanism of Vpu antagonizing the role of Tethin is still not completely clear at present. At first, it was reported that Vpu antagonized the mechanism of Tethin and the degradation of CD4 receptor, that is, through the combination of membrane helix region and Tethin, Tethin itself was degraded by ubiquitination enzyme and proteome pathway, so that it could not complete the physical system retention of the virus. However, subsequent studies confirmed that Vpu could also promote viral release without degrading, or reducing the amount of Tethin's expression on the surface of cells, suggesting that the theory was not complete. It was also reported that Tethin was able to specifically identify the viral replicon site, thus guessing that Vpu antagonized the role of Tethin to promote viral release by disrupting its recognition. There are some other reports showing that, in the presence of Vpu, Tethn can still be packaged into the progeny virus, but it is unable to play a mooring function, and the key molecular mechanism of Vpu antagonizing Tethin remains to be further elucidated. Vpu's own homologous oligomer forms a weakly selective potassium ion channel while Vpu can also interfere with TASK leading to depolarization of the host cell membrane, and we suspect that this depolarization phenomenon can trigger a voltage-dependent calcium ion channel, causing a rise in calcium ion concentration in the cell, This phenomenon has been preliminarily confirmed in previous studies. On the basis of this, we further confirm that Vpu causes intracellular calcium ion concentration to rise as a result of which voltage-dependent calcium ion channels trigger the correlation between this effect and its own ion channel activity. and the action of the relevant downstream pathway under the participation of calcium ions in the process of antagonizing Tethin by HIV-1Vpu, thereby providing a new idea for Vpu to antagonize Tethin. In this paper, we first explored the influence of Vpu on intracellular calcium ion concentration and the relationship with voltage-dependent calcium ion channels in 293T cells stably expressing Vpu. The calcium ions in cells were labeled with green fluorescent calcium indicator Fluo-4AM in the cells stably expressing Vpu, and the concentration of calcium ions in cells was detected by an inverted microscope, a spectrophotometer and a flow cytometer, and the up-regulation effect of Vpu on calcium ions in the cells was detected. At the same time, we detected the change of calcium ion concentration in the cells by means of VDCC antagonist and RNA silencing VDCC to determine that the change of calcium concentration is indeed by mediating the voltage-dependent calcium ion channel. Secondly, in this paper, we further study the influence of calcium ion as the downstream pathway of the medium on Vpu antagonist Tethin. We use the transmission electron microscope to observe whether Tethin has identified the change of membrane curvature of membrane membrane caused by sub-generation of virus by its special topological structure, and the interference of Vpu on the process and the effect of calcium ion on it; meanwhile, We also use Co-IP to verify whether Tethin and Gag are physically bonded to each other, and whether Vpu is affected by this process, and the results show that Vpu does not interfere with the process of Tethin to be located at the site. In addition, we tried to show that Vpu could inhibit the phosphorylation of Hem ITAM by increasing the intracellular calcium ion concentration, thus affecting the signal transduction activity of NF-Sepharose B and the role of calcium ion channels in a series of pathways. Finally, we further studied the possible mechanism of Vpu by activating the hydrolase to cut Tethin. The effect of calcium ions on the activity of matrix metalloenzymes and Furin enzymes and the effects of Tethin potential Furin cleavage site mutations on viral release were verified from gene levels and protein levels, and the results indicated that Furin did not cut Tethin, but it was interesting that Tethin could inhibit Furin's own activity. We have also made a preliminary inquiry about the potential cleavage of Tethin through the prediction of active site software. Through the above experiments, we examine the influence of Vpu on the concentration of calcium ions in the cells and the mechanism of downstream molecular mechanism related to the activity of Tethin, and provide a new direction for the further elucidation of the mechanism of Vpu antagonizing Tethin. In the future, we can comprehensively reveal the ways of Vpu-dependent calcium ion antagonizing Tethin.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R512.91
【相似文獻】
相關(guān)期刊論文 前10條
1 莊云詩;莊云龍;;用雙管鈣離子選擇性微電極測量大腦皮層鈣離子濃度的動態(tài)變化[J];化學(xué)傳感器;1986年04期
2 莊云龍;漆德瑤;戚挺華;高福鴻;;鈣離子選擇性微電極對體液及線粒體懸液中鈣離子的測定[J];化學(xué)傳感器;1990年02期
3 吳濤;鈣離子在細胞及迷路水平的生理作用[J];國外醫(yī)學(xué).耳鼻咽喉科學(xué)分冊;1997年04期
4 郭鴻涌;鄭末;邢凌霄;樊長在;張祥宏;顏威利;;工頻磁場對離體細胞胞漿鈣離子濃度的影響[J];河北大學(xué)學(xué)報(自然科學(xué)版);2008年01期
5 周永軍;牛中奇;侯建強;陳建華;白冰;黃華;;細胞內(nèi)自由鈣離子濃度變化的時-頻分析[J];中國生物醫(yī)學(xué)工程學(xué)報;2009年06期
6 莫鏡;崔其亮;張費通;林黎黎;李穎;廖嘉儀;;應(yīng)用激光共聚焦掃描顯微鏡技術(shù)檢測缺氧人肺微血管內(nèi)皮細胞內(nèi)鈣離子濃度動態(tài)變化[J];現(xiàn)代生物醫(yī)學(xué)進展;2011年05期
7 林金華;周艷芳;凌麗燕;姜寵華;陸衛(wèi)良;;頭孢曲松鈉與不同濃度含鈣溶液混合后鈣離子濃度的變化研究[J];中國藥房;2013年09期
8 關(guān)超然,龔明萃;熒光鈣離子指示劑在活細胞鈣利用研究中的應(yīng)用[J];基礎(chǔ)醫(yī)學(xué)與臨床;1990年01期
9 曹芳敏,楊柏松,唐曉賓;43名漢哈族兒童血中鈣離子濃度對比分析[J];臨床兒科雜志;2002年12期
10 趙樹仲;鈣離子在神經(jīng)介質(zhì)釋放中的作用[J];西安交通大學(xué)學(xué)報(醫(yī)學(xué)版);1981年03期
相關(guān)會議論文 前10條
1 應(yīng)陽君;;細胞質(zhì)鈣離子濃度非線性振蕩的一種同步機制分析[A];中國工程物理研究院科技年報（2001）[C];2001年
2 牛中奇;周永軍;侯建強;陳建華;白冰;黃華;;細胞內(nèi)自由鈣離子濃度變化的時-頻分析[A];中國儀器儀表學(xué)會醫(yī)療儀器分會第四次全國會員代表大會暨2009年學(xué)術(shù)年會論文集[C];2009年
3 郭義;張艷軍;陳靜子;徐湯蘋;陳波;;鈣離子是針刺起效的關(guān)鍵因素之一[A];第九屆全國鈣信號和細胞功能研討會論文摘要集[C];2012年
4 牛中奇;陳建華;侯建強;周永軍;王海彬;童穎;;細胞胞漿內(nèi)鈣離子濃度變化的時-頻分析[A];第十次中國生物物理學(xué)術(shù)大會論文摘要集[C];2006年
5 時靜華;陳曉東;潘桂蘭;;鈣離子在飲酒致心腦功能損傷中的作用[A];提高全民科學(xué)素質(zhì)、建設(shè)創(chuàng)新型國家——2006中國科協(xié)年會論文集[C];2006年
6 梁淑俊;鄭曉東;唐曉波;朱大嶺;;鈣離子在15-烷基二十碳四烯酸介導(dǎo)的肺動脈平滑肌收縮中的作用[A];中國藥理學(xué)會第九次全國會員代表大會暨全國藥理學(xué)術(shù)會議論文集[C];2007年
7 安少鋒;凌均h，
本文編號：2312044
本文鏈接：http://sikaile.net/yixuelunwen/nfm/2312044.html
最近更新
教材專著
