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

小肠结肠炎耶尔森氏菌和假结核耶尔森氏菌的致病性依赖于70 kb毒力质粒pYV，其编码复杂的III型分泌机制（YsC）以及几种Yop（耶尔森氏菌外蛋白）效应蛋白。效应蛋白之一是YopT，它通过III型分泌直接递送到靶细胞中。在真核细胞中，YopT引起Rho GTP酶的修饰，从而重新分布肌动蛋白细胞骨架。YopT是一种半胱氨酸蛋白酶，其在Rho蛋白的C-末端半胱氨酸之前连同结合的异戊二烯基团一起切割Rho蛋白。因此，GTdR从膜中释放。本研究的总体目标是研究YopT的动力学，并分析YopT识别的底物特异性和结构要求。此外，我们还希望对该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）在生物化学和功能上表征YopT的蛋白酶活性，并了解蛋白酶活性是如何调节的。因此，我们计划使用异戊二烯化和甲基化肽和HPLC分析建立定量体外测定。(ii)以研究底物特异性和由此产生的生物学后果。在第一步中，我们想要用YopT孵育肽，并通过MALDI-TOF质谱法分析切割。从这些数据出发，我们计划在体内研究确定的底物，并分析受影响的途径。(iii)确定YopT的关键结构特征，并鉴定体外和体内蛋白酶活性的最小必需结构，（iv）阐明YopT随后易位到靶细胞中的定位和相互作用。(v)计划研究YopT对RhoGT 3依赖性转录激活的影响。

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.