Mode of action of Yersinia enterocolitica "outer protein" T (Yop T)

小肠结肠炎耶尔森氏菌“外蛋白”T (Yop T) 的作用方式

• 批准号: 5414273
• 负责人: Professorin Dr. Gudula Schmidt
• 金额: --
• 依托单位: Abteilung I
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2003
• 资助国家: 德国
• 起止时间: 2002-12-31 至 2007-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5414273?language=en
• 关键词: Mode; action; Yersinia; enterocolitica; outer

The pathogenicity of Yersinia enterocolitica and Yersina pseudotuberculosis depends on a 70 kb virulence plasmid pYV, which encodes a complex type-III secretion machinery (YsC) as well as several Yop (Yersinia outer proteins) effector proteins. One of the effector proteins is YopT, which is directly delivered into the target cell by type-III secretion. In eukaryotic cells, YopT causes modification of Rho GTPases and thereby redistribution of the actin cytoskeleton. YopT is a cysteine protease, which cleaves Rho proteins in front of its C-terminal cysteine together with the bound isoprene groups. Thus the GTPase is released from membranes. The overall objective of this research is to study the kinetics of YopT and to analyze the substrate specificity and structural requirements for recognition by YopT. Moreover, we want to give a precise structure-function analyses of the protease. To achieve this goal, it will be necessary (i) to characterize the protease activity of YopT biochemically and functionally, and to understand how the protease activity is regulated. Therefore, we plan to establish a quantitative in vitro assay using isoprenylated and methylated peptides and HPLC analysis. (ii) to study the substrate specificity and the resulting biological consequences. In a first step we want to incubate peptides with YopT and analyze the cleavage by MALDI-TOF mass spectrometry. Proceeding from these data, we plan to study identified substrates in vivo and to analyzed the pathways affected. (iii) to determine crucial structural features of YopT and to identify the minimal essentiel structure for protease activity in vitro and in vivo, (iv) to clarify localization and interactions of YopT subsequent translocation into the target cells. (v) it is planned to study the influence of YopT on Rho GTPase dependent activation of transcription.

小肠结肠炎耶尔森氏菌和假结核耶尔森氏菌的致病性取决于一个70kb的毒力质粒pYV， pYV编码一个复杂的iii型分泌机制（YsC）以及几个Yop（耶尔森氏菌外蛋白）效应蛋白。其中一种效应蛋白是YopT，它通过iii型分泌直接进入靶细胞。在真核细胞中，YopT引起Rho gtpase的修饰，从而引起肌动蛋白细胞骨架的重新分配。YopT是一种半胱氨酸蛋白酶，它在其c端半胱氨酸和结合的异戊二烯基团前面切割Rho蛋白。因此，GTPase从膜中释放出来。本研究的总体目标是研究YopT的动力学，分析YopT识别的底物特异性和结构要求。此外，我们希望对蛋白酶进行精确的结构-功能分析。为了实现这一目标，有必要(i)从生化和功能上表征YopT的蛋白酶活性，并了解蛋白酶活性是如何被调节的。因此，我们计划建立一种使用异戊烯酰化和甲基化肽和高效液相色谱分析的体外定量分析方法。（ii）研究底物特异性和由此产生的生物学后果。在第一步，我们想用YopT孵育肽，并通过MALDI-TOF质谱分析裂解。从这些数据出发，我们计划在体内研究已确定的底物并分析受影响的途径。（iii）确定YopT的关键结构特征，并确定体外和体内蛋白酶活性的最小基本结构，（iv）阐明YopT在转运到靶细胞后的定位和相互作用。(v)计划研究YopT对Rho GTPase依赖性转录激活的影响。