Biochemical Characterization of Yersinia Effector YopJ.

描述（由申请人提供）：微生物病原体鼠疫耶尔森氏菌是造成欧洲、印度、中国甚至美国数百万人死亡的毁灭性瘟疫的病原体。这种病原体在几乎所有大陆的跳蚤/啮齿动物生态系统中持续存在，可导致未经治疗的受害者短暂但极其痛苦的死亡。这种病原体是一种可能用于生物战的潜在武器，因此了解这种毒素的工作原理非常重要。耶尔森菌的工作方式包括逃避吞噬、破坏宿主防御系统和诱导靶宿主细胞的程序性细胞死亡。只有6种被称为耶尔森氏菌外蛋白（Yops）的蛋白质编码在毒力质粒上有效地进行这些活动；其中之一的YopJ，既能阻断细胞因子的产生，又能促进靶宿主细胞的凋亡。YopJ是一种32kD蛋白，可阻断所有参与细胞因子产生的主要信号通路，包括所有MAPK信号通路和NFKappaB信号通路。YopJ蛋白家族由来自植物和动物病原体的效应物组成，与包括腺病毒蛋白酶和泛素样蛋白蛋白酶在内的半胱氨酸蛋白酶家族具有结构同源性。与YopJ是一种水解酶的提议一致，YopJ需要一个完整的催化位点来阻止进化上保守的MAPK激酶超家族（MKK）的激活。基于这些观察结果，我们提出YopJ使用一种新机制来破坏信号机制的一个组成部分，该组成部分是上述所有激酶驱动的信号级联反应的活性所必需的。我们提出了三个具体目标来研究我们的假设：(i)表征哺乳动物MAPK信号通路中耶尔森菌效应物YopJ的靶点。（ii）表征耶尔森菌效应物YopJ在NFkappaB信号通路中的靶标。（iii）表征YopJ的体外水解活性。这些研究的结果将确定YopJ的靶点，并表征YopJ的抑制活性，这将导致对细胞内信号传递所必需的进化保守调节机制的理解。这一机制的发现对于理解植物和动物的信号通路是如何调节的至关重要。

DESCRIPTION (provided by applicant): The microbial pathogen Yersinia pestis, was the causal agent for the devastating plagues killing millions of people in Europe, India, China and even the United States. The pathogen persists in flea/rodent ecosystems on almost every continent and can cause a short but extremely painful death in untreated victims. This pathogen is a potential weapon that may be used for Biowarfare and therefore it is important to understand how this toxin works. Yersinia's modus operandi includes evading phagocytosis, destroying the host defense system and inducing programmed cell death in the target host cell. Only six proteins called Yersinia outer proteins (Yops) encoded on a virulence plasmid efficiently carry out these activities; one of them, YopJ, both blocks cytokine production and promotes apoptosis in target host cells. YopJ, a 32kD protein, blocks all major signaling pathways involved in cytokine production including all the MAPK signaling pathways and the NFKappaB pathway. The family of YopJ proteins, composed of effectors from plant and animal pathogens, has structural homology to a clan of cysteine proteases that includes adenoviral proteases and the ubiquitin-like protein proteases. Consistent with the proposal that YopJ is a hydrolase, an intact catalytic site is required for YopJ to block the activation of the evolutionarily conserved super family of MAPK kinases (MKK). Based on these observations, we propose that YopJ uses a novel mechanism to disrupt a component of the signaling machinery that is required for the activity of all of the aforementioned kinase-driven signaling cascades. We propose three Specific Aims to investigate our hypothesis: (i) To characterize the target(s) of the Yersinia effector YopJ in the mammalian MAPK signaling pathway. (ii) To characterize the target(s) of the Yersinia effector YopJ in the NFkappaB signaling pathway. (iii) To characterize the hydrolytic activity of YopJ in vitro. Results from these studies will identify the targets of YopJ and characterize the inhibitory activity of YopJ that will lead to the understanding of an evolutionarily conserved mechanism of regulation that is necessary for intracellular transmission of signals. Discovery of this mechanism is essential for understanding how signaling pathways are regulated in both plants and animals.