鼠疫菌蛋白YpkA和人蛋白VASP的相互作用和功能研究

发布时间：2018-06-28 18:08

本文选题：YpkA + VASP　；参考：《中国人民解放军军事医学科学院》2011年博士论文

【摘要】：鼠疫的病原是鼠疫耶尔森氏菌(Yersinia pestis,简称鼠疫菌),分类上属于肠杆菌科耶尔森氏菌属。鼠疫菌是一种革兰氏阴性兼性胞内致病菌,一般通过染疫的蚤类叮咬在宿主动物间传播,偶尔通过接触染疫的动物皮毛、染疫的蚤类叮咬传播到人类。鼠疫菌拥有一个被称为III型分泌系统(Type III Secretion System,T3SS)的蛋白输送装置,它在抗吞噬、抑制细胞因子产生、促进宿主细胞凋亡等方面都发挥着极其重要的作用。通过它鼠疫菌将6个效应蛋白--YopH、YopE、YopT、YopM、YopJ/P和YpkA/YopO注射进入宿主细胞内,这些效应蛋白利用自身的生化活性所作用于不同的靶标,促进细菌感染的进行。其中一个效应蛋白YpkA(在小肠结肠炎耶尔森氏菌中称为YopO)是在原核生物中发现的第一个含有丝氨酸/苏氨酸激酶结构域的蛋白,由732aa组成,分子量为82KD,用有多个结构域。YpkA部分定位于细胞膜,能够引起细胞变圆,但是细胞并不脱落。YpkA的GDI结构域与宿主细胞RhoGDI在分子结构上非常相似,这使得YpkA能够模拟宿主的RhoGDI分子的功能,与RhoA/Rac1结合并导致它们的失活,从而破坏细胞骨架。尽管对YpkA已经有了一些研究,但是相对于YpkA复杂的结构和多样的功能,人们对其作用于细胞的分子机制仍然知之甚少。我们采用酵母双杂交的方法,以YpkA作为诱饵蛋白筛选人脾脏cDNA文库,获得了3个均指向VASP的阳性克隆,这说明YpkA很有可能与VASP相互作用。VASP(Vasodilator-Stimulated Phosphoprotein)属于Ena/VASP蛋白家族,是一种细胞骨架调节蛋白,在维持细胞形态和调节细胞运动中起重要作用。它能够直接促进肌动蛋白丝的组装,调节细胞层状伪足和片状伪足形成,从而起到影响细胞运动的作用。VASP的功能受到蛋白激酶的调节,它有三个磷酸化位点,即Ser157、Ser239和Thr278。这三个位点分别能够被PKA、PKG和AMPK特异性的磷酸化,VASP的磷酸化影响了它与其它蛋白的直接相互作用,进而影响了它自身及下游蛋白的功能,从而使它对肌动蛋白组装、应力纤维形成和细胞吞噬等过程的调节作用受到影响。可以看出,不论是从蛋白定位、生化基础,还从已知的功能来看,YpkA都存在着与VASP相互作用的基础。于是我们采用GST-Pull Down、免疫共沉淀和荧光共定位等技术在体内、体外验证YpkA与VASP的相互作用,确认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对VASP磷酸化的影响,采用定点突变技术研究对VASP的磷酸化是否具有位点特异性或优先性,以及这种磷酸化在VASP发挥功能中的作用。最后,采用报告基因、免疫荧光和RNAi等技术研究YpkA和VASP的相互作用对细胞F-actin组装、应力纤维形成和吞噬能力的影响。通过实验我们可以得出以下结论:鼠疫菌通过分泌的YpkA作用于VASP,促进VASP的磷酸化,抑制细胞内的肌动蛋白组装,破坏细胞应力纤维的形成,从而抑制细胞的吞噬功能,促进鼠疫菌在胞外的生存。这说明鼠疫菌在长期进化过程中具备了精巧的分子机制,以促进对机体的感染和致病。本研究揭示了耶尔森氏菌重要毒力因子YpkA在耶尔森氏菌和宿主细胞相互作用中所扮演的角色,促进对耶尔森氏菌致病机理的理解,为我们深入理解鼠疫菌致病机理提供了一个新角度,也为我们预防和治疗鼠疫提供了一个新的分子靶标。另外,本实验室使用酵母双杂交的方法对鼠疫菌蛋白和人蛋白之间相互作用进行了筛选。采用154个鼠疫菌毒力相关基因筛选了人脾脏cDNA文库,共获得了67个鼠疫菌蛋白和109个人蛋白之间的208对相互作用,根据这208对相互作用我们构建了一个鼠疫菌蛋白和人蛋白之间的相互作用网络。酵母双杂交作为目前大规模筛选蛋白-蛋白相互作用最可靠工具之一,已经被全世界的科学家广泛地用于筛选物种内部或物种之间的蛋白-蛋白相互作用,但是由于酵母双杂交技术本身存在的缺陷,很容易出现假阳性或加阴性。为了克服假阳性问题,我们采用GST Pull down的方法进行验证,总共验证了42对相互作用,其中37对阳性。由于发现多个鼠疫菌基因与NF-κB信号通路和MAPK信号通路分子存在相互作用,而NF-κB和MAPK报告基因活性检测起来又比较方便,我们又采用检测报告基因的方法验证这些鼠疫菌基因和免疫信号通路之间的相互作用。采用GST Pull down方法和报告基因检测这两种方法,对酵母双杂交获得的数据进行了蛋白-蛋白相互作用的物理性和功能性验证。根据这些对相互作用的验证,我们认为酵母双杂交得到的蛋白-蛋白相互作用数据还是可信度比较高的,可以对我们构建的网络进行下一步分析。分析鼠疫菌蛋白和人蛋白相互作用网络有助于我们深入理解鼠疫菌的致病机理和人体抗感染反应特征,为寻找针对鼠疫更好的疫苗和治疗靶标打下基础。
[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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R378

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