Identification of the non-proteolytic mechanism of NLRP1 activation

 描述(申请人提供)：炎性小体是一种先天免疫感知途径，旨在通过产生促炎介质和嗜热性细胞死亡来识别和清除感染性病原体。选择性靶向炎性小体及其传感器NLRPs正在成为疫苗学、肿瘤治疗和自身免疫治疗中调节免疫反应的重要手段。然而，到目前为止，NLRP激活的分子机制只有两种：炭疽杆菌致死因子对NLRP1的蛋白分解和NAIPS/NLRC4炎症体与鞭毛蛋白的结合。我最近确定，NLRP1还可以驱动宿主保护性炎症小体对原虫寄生虫弓形虫的反应。然而，与先前描述的由炭疽激活NLRP1的机制不同，NLRP1不是在弓形虫感染的反应中被蛋白质降解处理的，这导致了NLRP1已经进化到通过一种新的、未描述的机制来检测寄生虫感染的假设。这项K22奖将提供实验资源、时间和培训，以确定NLRP1传感途径的关键组成部分。 具体地说，这个奖项将使我能够发展1)设计和实施正向基因筛查的技能，2)寄生虫反向基因工程的工具和专业知识，3)探索NLRP1传感器激活所需组件的生化工具。这些研究涉及免疫学中一个研究不足的领域：真核病原体是如何被先天免疫系统感知的。此外，通过同时利用NLRP1的自然等位基因变异和寄生虫遗传学方法，我将建立一套独特的工具来定义NLRP1激活的分子基础，这将对我们理解炎症体生物学具有广泛的重要意义。具体地说，该项目的中心目标是1)确定触发NLRP1激活的寄生虫成分，2)确定NLRP1中介导这一反应的关键残基。

 DESCRIPTION (provided by applicant): Inflammasomes are innate immune sensing pathways designed to identify and clear infectious agents through production of pro-inflammatory mediators and pyroptotic cell death. Selective targeting of inflammasomes and their sensors, the NLRPs, is emerging as an important means of modulating the immune response in vaccinology, tumor therapy and treatment of autoimmunity. However, only two molecular mechanisms of NLRP activation have been described to date: NLRP1 proteolysis by Bacillus anthracis lethal factor and flagellin binding by the NAIPs/NLRC4 inflammasome. I have recently determined that NLRP1 also drives a host-protective inflammasome response to the protozoan parasite Toxoplasma gondii. In contrast to the previously described mechanism of NLRP1 activation by anthrax, however, NLRP1 is not proteolytically processed in response to Toxoplasma infection, leading to the hypothesis that NLRP1 has evolved to detect parasite infection via a novel, undescribed mechanism. This K22 award will provide the experimental resources, time and training to identify the critical components of the NLRP1 sensing pathway. Specifically, this award will allow me to develop 1) skills in the design and implementation of forward genetic screens, 2) tools and expertise in reverse genetic engineering of parasites, 3) biochemical tools to probe the essential components of NLRP1 for sensor activation. These studies address an understudied area in immunology: how eukaryotic pathogens are sensed by the innate immune system. Moreover, by exploiting the natural allelic variation in NLRP1 in parallel with a parasite genetics approach I will establish a unique set of tools to define the molecular underpinnings of NLRP1 activation that will be broadly important to our understanding of inflammasome biology. Specifically, the central goals of this project are to 1) identify the parasite components that trigger NLRP1 activation and 2) determine the critical residues in NLRP1 that mediate this response.