Activation mechanism of a NLR protein innate immune receptor

描述（由申请人提供）：监测微生物入侵信号通常是建立免疫反应的第一步。然而，我们对影响免疫力的系统和机制缺乏充分的了解。我的长期目标是了解驱动细胞内“传感器”蛋白激活的过程，这些蛋白可以识别微生物分子。这些细胞内受体属于核苷酸结合域（NBD）富含亮氨酸重复序列（LRR）蛋白家族（一般称为NLRs），是先天免疫系统的重要组成部分。这类基因的突变与人类疾病有关。考虑到它们对人类健康和作物生产力的重要性，我们目前对NLR蛋白在分子水平上的功能的了解是初步的。模式生物和人类生物学之间的知识转移加速了重大发现，因此理解NLR的功能和激活将需要一个协调的跨学科方法。我将使用一个典型的植物NLR，抗黄斑假单胞菌1 （RPM1）作为NLR激活的功能模型。RPM1通过效应诱导RPM1-相互作用4 （RIN4）蛋白磷酸化，间接识别两种不相关的致病菌III型效应蛋白（AvrB和AvrRpm1）。这一识别事件激活了一个复杂的输出反应，导致感染部位的程序性细胞死亡和病原体定植的限制。我们实验室最近发表和未发表的数据表明，与未磷酸化的RIN4相比，磷酸化的RIN4与RPM1具有更大的亲和力。我假设效应剂诱导的RIN4磷酸化增强了RPM1的n端线圈（CC）结构域和RIN4之间的静电相互作用，从而导致RPM1的构象变化，从而减轻RPM1 CC和LLR结构域施加的自抑制。这使得核苷酸交换/水解和随后的下游免疫信号传导成为可能。我的短期目标是测试我提出的模型，并使用本建议中强调的遗传和生化方法的混合物检查RPM1激活的物理属性。拟议的研究将阐明一个鲜为人知的NLR激活过程。

DESCRIPTION (provided by applicant): Monitoring signals of microbial invasion is universally the first step in mounting an immune response. Yet we lack a full understanding of the systems and mechanisms that influence immunity. My long term goal seeks to understand the processes that drive the activation of intracellular "sensor" proteins that recognize microbial molecules. These intracellular receptors belong to the nucleotide-binding domain (NBD) leucine-rich repeat (LRR) protein family (generically termed NLRs), and are critical components of innate immune systems. Mutations in this class of genes have been implicated in human disease. Considering their importance across kingdoms for human health and crop productivity our current understanding of NLR protein function at the molecular level is rudimentary. Knowledge transfer between model organisms and human biology has accelerated prominent discoveries and thus understanding NLR function and activation will require a concerted interdisciplinary approach. I will use a prototypical plant NLR, Resistance to Pseudomonas maculicola 1 (RPM1) as a functional model for NLR activation. RPM1 indirectly recognizes two unrelated pathogenic bacterial type III effector proteins (AvrB and AvrRpm1) via effector-induced phosphorylation of the RPM1- interacting 4 (RIN4) protein. This recognition event activates a complex output response, resulting in programmed cell death at the infection site and restriction of pathogen colonization. Recent published and unpublished data from our laboratory suggests phosphorylated RIN4 associates with RPM1 with a greater affinity than does unphosphorylated RIN4. I hypothesize effector-induced phosphorylation of RIN4 enhances electrostatic interactions between the N-terminal coiled-coil (CC) domain of RPM1 and RIN4 entailing a stable interaction and leading to a RPM1 conformational change that relieves auto-inhibition imposed by the RPM1 CC and LLR domains. This enables nucleotide exchange/hydrolysis and subsequent downstream immune signaling. My short term goals are to test my proposed model and examine the physical attributes of RPM1 activation using a mixture of genetic and biochemical approaches highlighted in this proposal. The proposed research will shed light on a poorly understood NLR activation process.