Elucidating the Structural Mechanisms of NAIP Receptors in Bacterial Detection and Inflammasome Activation

项目摘要 炎性小体是一种巨道尔顿蛋白复合物，它启动炎症反应， 在先天免疫系统中的作用。一旦激活，炎性小体募集并激活效应蛋白 半胱天冬酶-1。半胱天冬酶-1又切割Gasdermin D（GSDMD）蛋白，释放其N-末端结构域， 插入细胞膜在细胞表面打孔。结果，宿主细胞将经历热凋亡， 细胞死亡（焦亡）并将细胞内容物释放到细胞外环境中。促炎 细胞因子IL-1 β和IL-18，两者都被caspase-1激活，也将被释放以激活下游的 炎症反应。 NAIP是细胞溶质免疫受体的家族，其激活细胞中的NAIP/NLRC 4炎性体。 对革兰氏阴性细菌感染的反应。在小鼠中有7种NAIP蛋白，每种蛋白都有一个特异性的 III型分泌系统中的鞭毛蛋白、针蛋白或内杆蛋白等配体。在以前的研究中， 我们已经表明活性NAIP通过成核聚合机理活化NLRC 4。但据 关于NAIP如何在静息细胞中保持无活性，以及配体特异性如何在静息细胞中保持无活性， 在不同的NAIP中确定。与小鼠不同，人类只有一个NAIP， 被所有三种细菌配体激活这就引出了许多问题，包括什么是结构基础， 通过人类NAIP广泛检测细菌配体，它告诉我们关于人类炎性小体的什么？ 细菌检测机制？为了解决这些问题，我们建议将联合收割机电子冷冻显微镜 （cryoEM）与生物化学和功能研究，以阐明信号的结构机制 通过NAIP蛋白转导。NAIP/NLRC 4炎性小体的失调导致巨噬细胞活化 NAIP蛋白的突变与脊髓肌萎缩症（MAS）和自身炎症密切相关， 萎缩（SMA）。这项工作的成功执行将广泛推动有效疗法的发展 通过靶向炎性小体治疗感染性疾病、自身炎性疾病、SMA和癌症 通路

Project Summary Inflammasomes are mega-Dalton protein complexes that initiate inflammation responses and play important roles in the innate immune system. Upon activation, inflammasomes recruit and activate the effector protein caspase-1. Caspase-1 in turn cleaves the Gasdermin D (GSDMD) protein to release its N-terminal domain, which inserts into the cell membrane to punch holes on the cell surface. As a result, the host cell will undergo pyroptotic cell death (pyroptosis) and release the cell contents into the extracellular environment. Pro-inflammatory cytokines IL-1 and IL-18, both activated by caspase-1, will also be released to activate the downstream inflammation reactions. NAIP is a family of cytosolic immunological receptors that activate the NAIP/NLRC4 inflammasomes in response to Gram-negative bacterial infections. There are seven NAIP proteins in mouse, each sense a specific ligand such as Flagellin, Needle protein or Inner Rod protein in the type III secretion system. In previous studies, we have shown active NAIPs activate NLRC4 through the nucleated polymerization mechanism. However, it is still largely unknown about how NAIPs remain inactive in the resting cells, and how is the ligand specificity is determined among different NAIPs. Unlike in mouse, humans only have one NAIP and it was shown to be activated by all three bacterial ligands. This leads to many questions including what is the structural basis of the broad detection of bacterial ligands by human NAIP and what does it tell us about the human inflammasomal bacterial detection mechanism? To address these questions, we propose to combine electron cryo-microscopy (cryoEM) with biochemical and functional studies to elucidate the structural mechanisms underlying the signal transduction by NAIP proteins. Dysregulation of NAIP/NLRC4 inflammasomes causes macrophage activation syndrome (MAS) and autoinflammation, and mutations of NAIP protein are highly correlated with spinal muscular atrophy (SMA). The successful execution of this work will broadly advance the development of effective therapies to treat infectious diseases, autoinflammatory diseases, SMA, and cancer through targeting the inflammasome pathway.