本文選題：YpkA + VASP　； 參考：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2011年博士論文

【摘要】：鼠疫的病原是鼠疫耶爾森氏菌(Yersinia pestis,簡(jiǎn)稱鼠疫菌),分類上屬于腸桿菌科耶爾森氏菌屬。鼠疫菌是一種革蘭氏陰性兼性胞內(nèi)致病菌,一般通過染疫的蚤類叮咬在宿主動(dòng)物間傳播,偶爾通過接觸染疫的動(dòng)物皮毛、染疫的蚤類叮咬傳播到人類。鼠疫菌擁有一個(gè)被稱為III型分泌系統(tǒng)(Type III Secretion System,T3SS)的蛋白輸送裝置,它在抗吞噬、抑制細(xì)胞因子產(chǎn)生、促進(jìn)宿主細(xì)胞凋亡等方面都發(fā)揮著極其重要的作用。通過它鼠疫菌將6個(gè)效應(yīng)蛋白--YopH、YopE、YopT、YopM、YopJ/P和YpkA/YopO注射進(jìn)入宿主細(xì)胞內(nèi),這些效應(yīng)蛋白利用自身的生化活性所作用于不同的靶標(biāo),促進(jìn)細(xì)菌感染的進(jìn)行。其中一個(gè)效應(yīng)蛋白YpkA(在小腸結(jié)腸炎耶爾森氏菌中稱為YopO)是在原核生物中發(fā)現(xiàn)的第一個(gè)含有絲氨酸/蘇氨酸激酶結(jié)構(gòu)域的蛋白,由732aa組成,分子量為82KD,用有多個(gè)結(jié)構(gòu)域。YpkA部分定位于細(xì)胞膜,能夠引起細(xì)胞變圓,但是細(xì)胞并不脫落。YpkA的GDI結(jié)構(gòu)域與宿主細(xì)胞RhoGDI在分子結(jié)構(gòu)上非常相似,這使得YpkA能夠模擬宿主的RhoGDI分子的功能,與RhoA/Rac1結(jié)合并導(dǎo)致它們的失活,從而破壞細(xì)胞骨架。盡管對(duì)YpkA已經(jīng)有了一些研究,但是相對(duì)于YpkA復(fù)雜的結(jié)構(gòu)和多樣的功能,人們對(duì)其作用于細(xì)胞的分子機(jī)制仍然知之甚少。 我們采用酵母雙雜交的方法,以YpkA作為誘餌蛋白篩選人脾臟cDNA文庫,獲得了3個(gè)均指向VASP的陽性克隆,這說明YpkA很有可能與VASP相互作用。VASP(Vasodilator-Stimulated Phosphoprotein)屬于Ena/VASP蛋白家族,是一種細(xì)胞骨架調(diào)節(jié)蛋白,在維持細(xì)胞形態(tài)和調(diào)節(jié)細(xì)胞運(yùn)動(dòng)中起重要作用。它能夠直接促進(jìn)肌動(dòng)蛋白絲的組裝,調(diào)節(jié)細(xì)胞層狀偽足和片狀偽足形成,從而起到影響細(xì)胞運(yùn)動(dòng)的作用。VASP的功能受到蛋白激酶的調(diào)節(jié),它有三個(gè)磷酸化位點(diǎn),即Ser157、Ser239和Thr278。這三個(gè)位點(diǎn)分別能夠被PKA、PKG和AMPK特異性的磷酸化,VASP的磷酸化影響了它與其它蛋白的直接相互作用,進(jìn)而影響了它自身及下游蛋白的功能,從而使它對(duì)肌動(dòng)蛋白組裝、應(yīng)力纖維形成和細(xì)胞吞噬等過程的調(diào)節(jié)作用受到影響。 可以看出,不論是從蛋白定位、生化基礎(chǔ),還從已知的功能來看,YpkA都存在著與VASP相互作用的基礎(chǔ)。于是我們采用GST-Pull Down、免疫共沉淀和熒光共定位等技術(shù)在體內(nèi)、體外驗(yàn)證YpkA與VASP的相互作用,確認(rèn)YpkA和VASP的相互作用不是物理性的,而是功能性的。然后研究YpkA對(duì)VASP磷酸化的影響,采用定點(diǎn)突變技術(shù)研究對(duì)VASP的磷酸化是否具有位點(diǎn)特異性或優(yōu)先性,以及這種磷酸化在VASP發(fā)揮功能中的作用。最后,采用報(bào)告基因、免疫熒光和RNAi等技術(shù)研究YpkA和VASP的相互作用對(duì)細(xì)胞F-actin組裝、應(yīng)力纖維形成和吞噬能力的影響。通過實(shí)驗(yàn)我們可以得出以下結(jié)論:鼠疫菌通過分泌的YpkA作用于VASP,促進(jìn)VASP的磷酸化,抑制細(xì)胞內(nèi)的肌動(dòng)蛋白組裝,破壞細(xì)胞應(yīng)力纖維的形成,從而抑制細(xì)胞的吞噬功能,促進(jìn)鼠疫菌在胞外的生存。這說明鼠疫菌在長(zhǎng)期進(jìn)化過程中具備了精巧的分子機(jī)制,以促進(jìn)對(duì)機(jī)體的感染和致病。本研究揭示了耶爾森氏菌重要毒力因子YpkA在耶爾森氏菌和宿主細(xì)胞相互作用中所扮演的角色,促進(jìn)對(duì)耶爾森氏菌致病機(jī)理的理解,為我們深入理解鼠疫菌致病機(jī)理提供了一個(gè)新角度,也為我們預(yù)防和治療鼠疫提供了一個(gè)新的分子靶標(biāo)。 另外,本實(shí)驗(yàn)室使用酵母雙雜交的方法對(duì)鼠疫菌蛋白和人蛋白之間相互作用進(jìn)行了篩選。采用154個(gè)鼠疫菌毒力相關(guān)基因篩選了人脾臟cDNA文庫,共獲得了67個(gè)鼠疫菌蛋白和109個(gè)人蛋白之間的208對(duì)相互作用,根據(jù)這208對(duì)相互作用我們構(gòu)建了一個(gè)鼠疫菌蛋白和人蛋白之間的相互作用網(wǎng)絡(luò)。酵母雙雜交作為目前大規(guī)模篩選蛋白-蛋白相互作用最可靠工具之一,已經(jīng)被全世界的科學(xué)家廣泛地用于篩選物種內(nèi)部或物種之間的蛋白-蛋白相互作用,但是由于酵母雙雜交技術(shù)本身存在的缺陷,很容易出現(xiàn)假陽性或加陰性。為了克服假陽性問題,我們采用GST Pull down的方法進(jìn)行驗(yàn)證,總共驗(yàn)證了42對(duì)相互作用,其中37對(duì)陽性。由于發(fā)現(xiàn)多個(gè)鼠疫菌基因與NF-κB信號(hào)通路和MAPK信號(hào)通路分子存在相互作用,而NF-κB和MAPK報(bào)告基因活性檢測(cè)起來又比較方便,我們又采用檢測(cè)報(bào)告基因的方法驗(yàn)證這些鼠疫菌基因和免疫信號(hào)通路之間的相互作用。采用GST Pull down方法和報(bào)告基因檢測(cè)這兩種方法,對(duì)酵母雙雜交獲得的數(shù)據(jù)進(jìn)行了蛋白-蛋白相互作用的物理性和功能性驗(yàn)證。根據(jù)這些對(duì)相互作用的驗(yàn)證,我們認(rèn)為酵母雙雜交得到的蛋白-蛋白相互作用數(shù)據(jù)還是可信度比較高的,可以對(duì)我們構(gòu)建的網(wǎng)絡(luò)進(jìn)行下一步分析。分析鼠疫菌蛋白和人蛋白相互作用網(wǎng)絡(luò)有助于我們深入理解鼠疫菌的致病機(jī)理和人體抗感染反應(yīng)特征,為尋找針對(duì)鼠疫更好的疫苗和治療靶標(biāo)打下基礎(chǔ)。
[Abstract]:The plague is the pathogen of the plague, Jerson Prand (Yersinia pestis, abbreviated as Yersinia), belonging to the genus Kyel Sen, a gram negative facultative pathogenic bacterium, usually transmitted by the bite of the flea infected by the epidemic, occasionally through the fur of animals exposed to epidemic disease, and the transmission of the flea bites of the epidemic. To human. Yersinia pestis has a protein delivery device called Type III Secretion System (T3SS), which plays an extremely important role in anti phagocytosis, inhibiting cytokine production and promoting apoptosis of host cells. Through it, the 6 Effect proteins --YopH, YopE, YopT, YopM, YopJ/P and YpkA/YopO are the 6 Effect proteins of Yersinia pestis. Injection into the host cells, these effect proteins use their own biochemical activity to different targets and promote bacterial infection. One of the effector protein YpkA (called YopO in Jerson S bacteria enterocolitis) is the first protein containing the serine / threonine kinase domain found in the prokaryotes, which is 73 2AA, which is composed of 82KD, is located in the cell membrane with multiple domains of.YpkA, which can cause the cell to circle, but the GDI domain of the.YpkA is very similar to the host cell RhoGDI in the molecular structure, which enables YpkA to mimic the function of the RhoGDI molecules in the host and to combine with RhoA/Rac1 and lead to their inactivation. It destroys the cytoskeleton. Although some studies have been made on YpkA, there is still little knowledge about the molecular mechanisms of the cells in relation to the complex structure and various functions of YpkA.
We use yeast two hybrid method to screen human spleen cDNA library with YpkA as bait protein. 3 positive clones pointing to VASP are obtained. This shows that YpkA is very likely to interact with VASP,.VASP (Vasodilator-Stimulated Phosphoprotein) belongs to the Ena/VASP protein family, is a cytoskeleton regulatory protein, in the maintenance of cell shape. State and regulation of cell movement play an important role. It can directly promote the assembly of actin filaments, regulate the formation of cell layer pseudo foot and plate-like pseudo foot, so that the function of.VASP is regulated by protein kinase, and it has three phosphorylation sites, that is, Ser157, Ser239 and Thr278., respectively. The phosphorylation of PKA, PKG and AMPK, the phosphorylation of VASP affects its direct interaction with other proteins, and then affects its own and downstream protein functions, thereby affecting the regulation of actin assembly, stress fiber formation and cell phagocytosis.
It can be seen that, in terms of protein location, biochemical basis, and known function, YpkA has the basis of interaction with VASP. So we used GST-Pull Down, immunoprecipitation and fluorescence co localization in vivo to verify the interaction between YpkA and VASP in vitro, and confirm that the interaction between YpkA and VASP is not physical, It is functional. Then we study the effect of YpkA on the phosphorylation of VASP, using site directed mutagenesis to study whether the phosphorylation of VASP has loci specificity or priority, and the role of this phosphorylation in the function of VASP. Finally, the interaction of YpkA and VASP on cell F is studied by using a reporter gene, immunofluorescence and RNAi. -actin assembly, the effect of stress fiber formation and phagocytosis. Through the experiment, we can draw the following conclusions: Yersinia pestis can promote the phosphorylation of VASP through the secretion of VASP, inhibit the assembly of actin in cell, destroy the formation of cell stress fiber, inhibit the phagocytosis of cell and promote the growth of Yersinia pestis in the extracellular. This shows that Yersinia pestis has a delicate molecular mechanism in the course of long-term evolution to promote infection and pathogenicity to the body. This study reveals the role of Jerson Prand's important virulence factor YpkA in the interaction between Jerson Prand and host cells, and promotes understanding of the pathogenesis of yersson bacteria, and provides us with a deep understanding of the pathogenic mechanism of Yersinia. It provides a new perspective for understanding the pathogenic mechanism of Yersinia pestis, and also provides a new molecular target for preventing and treating plague.
In addition, the interaction between Yersinia pestis protein and human protein was screened by yeast two hybrid method. The human spleen cDNA library was screened by 154 strains of Yersinia pestis virulence related genes, and a total of 67 plague bacteria proteins and 208 pairs of proteins were interacted with each other. The interaction between the 208 pairs of proteins was constructed. As one of the most reliable tools for large-scale screening of protein protein interactions, it has been widely used by scientists all over the world to screen protein protein interactions within or between species, but because of the yeast two hybrid technology itself, yeast two hybrid is one of the most reliable tools for screening protein protein interactions in large scale. In order to overcome the false positive problem, we used the GST Pull down method to verify the 42 pairs of interactions, including 37 positive. Because of the discovery of multiple Yersinia gene, the interaction between the NF- kappa B signaling pathway and the MAPK signaling pathway, and the NF- kappa B and MAPK reports The detection of gene activity is more convenient. We also use the method of detecting the reporter gene to verify the interaction between the Yersinia pestis gene and the immune signal pathway. The GST Pull down method and the report gene are used to detect these two methods, and the physical and work of the protein protein interaction are obtained by the data obtained by the yeast two hybrid. Verification. Based on the validation of these interactions, we think that the protein protein interaction data obtained by yeast two hybrid are still more reliable, and we can analyze the network we build next. The analysis of the protein and human protein interaction network of Yersinia pestis can help us to understand the pathogenesis of Yersinia pestis. And the characteristics of human anti infection response, laying the foundation for finding better vaccines and therapeutic targets against plague.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R378

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