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

项目摘要 NLRP 3（NOD样受体蛋白3）是一种细胞内传感器，其检测多种刺激，包括 感染和代谢功能障碍导致大分子信号传导复合物的组装， 炎性小体，其促进细胞因子IL-1b和IL-18的半胱天冬酶-1依赖性加工， 生物活性形式;这些细胞因子又是许多自身炎症，自身免疫， 代谢和传染病。NLRP 3被广泛的分子激活，包括 尿酸盐晶体，病毒RNA，甚至细菌的成孔毒素，但很少有人知道的机制， 其中NLRP 3感知并激发对这些化学和结构上不同的刺激的响应。我们和 其他人先前已经表明NLRP 3在激活时与线粒体结合。无论这个链接 简单地指出了对膜平台的要求，以实现高效的固相组装 炎症体信号传导组分或线粒体对NLRP 3活化的主动调节 组成部分，甚至线粒体蛋白质仍然是模糊的。目前还不清楚是否以及如何相互作用， NLRP 3与线粒体一起影响线粒体功能。我们之前已经表明，NLRP 3 线粒体募集和随后的炎性小体反应依赖于短N- NLRP 3的末端序列与外部线粒体衔接蛋白MAVS。然而，MAVS特别 响应于非结晶而非结晶NLRP 3活化剂而增强炎性小体组装 提示线粒体调节NLRP 3炎性体的有趣和复杂的机制 activation.在这项研究中，我们提出了额外的线粒体蛋白控制NLRP 3炎性体， 可以想象的是，以刺激特异性的方式响应于不同的激活剂的激活。响应于 RNA病毒的检测MAVS不仅与NLRP 3有关，而且与RIG-I样受体（RLR）有关， 产生1型干扰素，表明NLRP 3和RLR信号之间存在意外串扰 途径。在这个建议中，我们将阐明线粒体/MAVS调节NLRP 3的机制， 激活并检查其与RLR途径的串扰。这将通过以下方式实现：1）质量 光谱法鉴定与炎性小体复合物相关的其他线粒体蛋白， 对晶体和非晶体NLRP 3激活剂的反应，然后进行测定以评估它们对 炎性小体激活和线粒体功能，2）软X射线断层扫描和相关荧光 显微镜以评估炎性小体组装中对亚细胞膜平台的需求，以及3） 病毒感染的模型，以确定MAVS依赖的串扰之间的生理后果， NLRP 3和RLR通路。这些调查将产生重要的机械见解如何 线粒体和MAVS调节NLRP 3炎性小体活化，这可能与调节 NLRP 3活性不仅在病毒感染中，而且在多种代谢疾病中。

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.