NLRP3 inflammasome activation and its crosstalk with RLR signaling at the mitochondria

项目摘要 NLRP3(节点样受体蛋白3)是一种细胞内传感器，可以检测包括 感染和代谢障碍导致大分子信号复合体组装 炎症体促进caspase-1依赖的细胞因子IL-1b和IL-18的处理 生物活性形式；这些细胞因子反过来又是许多自体炎症、自身免疫、 代谢性和传染性疾病。NLRP3被包括单钠在内的多种分子激活 尿酸盐晶体，病毒RNA，甚至细菌致孔毒素，但对其作用机制知之甚少 其中NLRP3感知并对这些化学和结构上不同的刺激产生反应。我们和 此前已有研究表明，NLRP3在激活时与线粒体相关。此链接是否 简单地指出了对膜平台的要求，以实现高效的固相组装 炎症体信号成分或线粒体对NLRP3激活的主动调节 成分，甚至线粒体蛋白仍然含糊不清。也不清楚是否以及如何相互作用 带有线粒体的NLRP3影响线粒体功能。我们之前已经证明了NLRP3 线粒体募集和随后的炎症体反应依赖于短N- NLRP3的末端序列与线粒体外部适配蛋白MAVS。然而，小牛特别是 增加炎症小体对非晶态而不是晶态NLRP3激活剂的反应 提示线粒体调节NLRP3炎症体的有趣而复杂的机制 激活。在这项研究中，我们提出了额外的线粒体蛋白控制NLRP3炎症体 对不同的激活剂作出反应的激活，可以想象是以刺激特定的方式。作为对.的回应 RNA病毒MAV的检测不仅通过NLRP3，而且还通过RIG-I样受体(RLRs)来实现 产生1型干扰素，表明NLRP3和RLR信号之间存在意外的串扰 小路。在这个提案中，我们将阐明线粒体/MAV调节NLRP3的机制 激活并检测其与RLR通路的串扰。这将通过以下方式实现：1)质量 光谱分析鉴定与炎症体复合体相关的额外线粒体蛋白 对晶体和非晶态NLRP3激活剂的响应，然后进行检测以评估它们对 炎性小体激活与线粒体功能；2)软X射线断层扫描及相关荧光 显微镜评估炎性小体组装中对亚细胞膜平台的要求，以及3) 病毒感染模型，以确定MAV依赖的串扰在 NLRP3和RLR通路。这些调查将产生重要的机械性见解， 线粒体和MAV调节NLRP3炎症体的激活，这可能与调控有关 NLRP3活性的变化不仅在病毒感染中，而且在各种代谢性疾病中也是如此。

Project Summary NLRP3 (NOD-Like Receptor Protein 3) is an intracellular sensor that detects a variety of stimuli including infection and metabolic dysfunction resulting in assembly of a macromolecular signaling complex called the inflammasome which promotes caspase-1 dependent processing of the cytokines IL-1b and IL-18 to their bioactive forms; these cytokines in turn underlie the pathophysiology of many autoinflammatory, autoimmune, metabolic and infectious diseases. NLRP3 is activated by a wide-range of molecules including monosodium urate crystals, viral RNA and even bacterial pore-forming toxins, but little is known about the mechanisms by which NLRP3 senses and elicits a response to these chemically and structurally dissimilar stimuli. We and others have previously shown that NLRP3 associates with mitochondria upon activation. Whether this link simply points to a requirement for a membrane platform to achieve efficient solid-phase assembly of inflammasome signaling components or an active modulation of NLRP3 activation by mitochondrial components or even mitochondrial proteins remains ambiguous. It is also unclear if and how interaction of NLRP3 with mitochondria influences mitochondrial functions. We have previously shown that NLRP3 mitochondrial recruitment and the ensuing inflammasome response is dependent on interaction of a short N- terminal sequence in NLRP3 with the outer mitochondrial adapter protein MAVS. However, MAVS specifically augments inflammasome assembly in response to non-crystalline, but not crystalline NLRP3 activators suggesting an interesting and complex mechanism by which mitochondria regulate NLRP3 inflammasome activation. In this study, we propose that additional mitochondrial proteins control NLRP3 inflammasome activation in response to different activators, conceivably in a stimulus-specific manner. In response to detection of RNA viruses MAVS is engaged not only by NLRP3 but also by the RIG-I- like receptors (RLRs) to produce type 1 interferons, suggesting an unexpected crosstalk between the NLRP3 and RLR signaling pathways. In this proposal, we will elucidate the mechanisms by which mitochondria/MAVS regulate NLRP3 activation and examine its crosstalk with the RLR pathway. This will be accomplished by: 1) mass spectrometry to identify additional mitochondrial proteins that associate with inflammasome complexes in response to crystals and non-crystalline NLRP3 activators, followed by assays to evaluate their effect on inflammasome activation and mitochondrial functions, 2) soft X-ray tomography and correlative fluorescence microscopy to assess a requirement for subcellular membrane platforms in inflammasome assembly, and 3) models of viral infection to identify the physiological consequences of MAVS-dependent crosstalk between the NLRP3 and RLR pathways. These investigations will yield important mechanistic insights into how mitochondria and MAVS regulate NLRP3 inflammasome activation, which may be of relevance for modulation of NLRP3 activity not only in viral infections but also in a variety of metabolic diseases.