Functional and Structural Dissection of Inflammasome Activation

这是R37 AI 075039的MERIT扩展。在上一个融资期，我们解决了基本问题， 病原体编码的毒力活动被先天免疫系统检测的机制。 我们专注于一种称为NLRP 1炎性体的先天免疫传感器。这个传感器被认为是 由细菌病原体芽孢杆菌分泌的一种称为致死因子（LF）的蛋白酶毒素激活 炭疽病我们证明LF通过直接蛋白水解NLRP 1激活NLRP 1。我们发现 这种切割导致蛋白酶体对NLPR 1的N端规则依赖性降解。 然而，出乎意料的是，我们发现LF诱导的降解不会破坏NLRP 1。而是 发现NLRP 1经历了“功能性降解”，这是一个独特的过程， NLRP 1的C-末端CARD结构域被释放，从而能够进行寡聚化， 炎性小体形成。我们的工作是确定NLRP 1能够感知的分子基础， 病原体编码的酶活性使我们想知道NLRP 1是否可以感知其他病原体- 编码的活动，此外，是否可以检测到额外的病原体编码的效应物， 其他先天免疫传感器事实上，我们发现一种病原体编码的泛素E3连接酶， 由细菌病原体福氏志贺菌分泌到宿主细胞中的IpaH 7.8也被NLRP 1感测。在 在延长期内，我们建议继续研究 病原体编码的毒力因子的酶活性由先天免疫系统检测。我们 正在进行筛选，以确定刺激宿主免疫反应的新型毒力因子。我们将 并对这些毒力因子的感受器及其分子基础进行了机理研究 传感器激活。我们的研究将共同阐明基本的分子机制， 病原体被先天免疫系统感知。 相关性（参见说明）： 人类健康依赖于先天免疫系统对病原微生物的准确和灵敏的检测， 免疫系统我们建议确定毒力活动的基本机制， 细菌病原体被先天免疫系统感知。

This is a MERIT extension of R37 AI075039. In the previous funding period we addressed the fundamental mechanisms by which pathogen-encoded virulence activities are detected by the innate immune system. We focused on an innate immune sensor called the NLRP1 inflammasome. This sensor was known to be activated by a protease toxin, called lethal factor (LF), that is secreted by the bacterial pathogen Bacillus anthracis. We demonstrated that LF activates NLRP1 by direct proteolytic cleavage of NLRP1. We found that this cleavage results in the N-end rule-dependent degradation of NLPR1 by the proteasome. Unexpectedly, however, we found that LF-induced degradation does not inactivate NLRP1. Instead, we found that NLRP1 underwent “functional degradation”, a unique process by which the signaling competent C-terminal CARD domain of NLRP1 was released and thereby able to undergo oligomerization and inflammasome formation. Our work identifying the molecular basis by which NLRP1 is able to sense pathogen-encoded enzymatic activity led us to ask whether NLRP1 might sense additional pathogen- encoded activities and, moreover, whether additional pathogen-encoded effectors might be detected by other innate immune sensors. Indeed, we discovered that a pathogen-encoded ubiquitin E3 ligase called IpaH7.8, secreted into host cells by the bacterial pathogen Shigella flexneri, is also sensed by NLRP1. In the extension period, we propose to continue to investigate the molecular mechanisms by which the enzymatic activities of pathogen-encoded virulence factors are detected by the innate immune system. We are conducting screens to identify novel virulence factors that stimulate host immune responses. We will then conduct mechanistic studies to identify the sensors of these virulence factors and the molecular basis of sensor activation. Together our studies will illuminate fundamental molecular mechanisms by which pathogens are sensed by the innate immune system. RELEVANCE (See instructions): Human health depends on the accurate and sensitive detection of pathogenic microbes by the innate immune system. We propose to determine fundamental mechanisms by which the virulence activities of bacterial pathogens are sensed by the innate immune system.