Cell-free formation, visualization and study of inflammasomes in real-time with optical tweezers and confocal fluorescence microscopy

项目摘要/摘要。炎症是先天免疫系统对抗的主要反应 感染。它的失调会导致慢性炎症，这是威胁生命的疾病的主要原因。一开始 炎症依赖于被称为炎症体的多蛋白复合体在细胞内的组装， 它们通过三种蛋白质的多个拷贝的齐聚作用，在有害物质的存在下形成： 对病原体或受损组织发出的危险信号做出反应的传感器；-激活的proaspase-1 炎性细胞因子；-适配器ASC，通过连接传感器和 原天冬氨酸酶-1分子。研究支配炎症体组装的分子机制是 由于其动态的性质和炎症体相关蛋白对自身的强烈倾向而具有挑战性 助理。这些挑战限制了我们对炎性小体组装的理解。具体来说，AIM2-ASC 炎症小体作为AIM2感受器，在抵抗入侵病原体的先天免疫系统中起着至关重要的作用 能够检测到外来DNA。尽管对AIM2-ASC的运行模式进行了广泛的研究 炎症体，AIM2、ASC和DNA在炎症体形成过程中的相互作用还不完全清楚。 特别是，AIM2-ASC炎症小体组装所涉及的动力学参数是未知的，并且 关于AIM2调控的分子基础存在知识缺口。填补这些空白将有助于 确定操纵炎性小体的分子干预措施。单分子技术需要 亚纳摩尔蛋白浓度，从而使大量前炎性小体组装的动力学研究成为可能 寡聚化就会发生。特别是，光镊子可以利用AIM2-DNA结合，并允许完全机械 对单个DNA分子的控制。通过将光学镊子、共聚焦荧光显微镜结合在一起，实现了实时 时间可视化和微流控技术用于逐步添加炎症体成分，该建议旨在 在无细胞系统中形成并实时显示AIM2-ASC炎症小体。证据是 介绍了这一策略的应用，展示了AIM2低聚物随机结合的实时组装 被两个光学捕获的珠子捆绑在一起的单个DNA分子。这些有希望的成果将被用来 确定全长ASC和AIM2在炎性小体组装中的协同作用(目标1)并建立 AIM2天然抑制剂对炎症小体调节的作用机制(目标2)。最重要的是 该项目的意义在于获得有关炎性小体激活和调节机制的信息 通过使用拟议的战略克服目前的限制：a)提供关于动力学的未知信息 和AIM2-ASC炎症体的组装；b)为扩展到其他炎症体奠定基础 获取有关炎症体运行模式的全面相关数据；c)检测炎症体抑制剂 在分子水平上深入了解它们的功能；d)促进分子模型的设计 指由于炎性小体过度激活而引起的疾病。

PROJECT SUMMARY/ABSTRACT. Inflammation is the primary response of the innate immune system to fight infection. Its dysregulation leads to chronic inflammation, a major cause of life-threatening diseases. The onset of inflammation depends on the intracellular assembly of multiprotein complexes known as inflammasomes, which form upon the presence of harmful substances by oligomerization of multiple copies of three proteins: - sensors that react upon danger signals derived from pathogens or damaged tissue; - procaspase-1 that activates inflammatory cytokines; - the adaptor ASC that functions as a molecular glue by connecting sensor and procaspase-1 molecules. Studying the molecular mechanisms that govern inflammasome assembly is challenging due to its dynamic nature and the strong tendency of inflammasome-related proteins to self- associate. These challenges limit our understanding of inflammasome assembly. Specifically, the AIM2-ASC inflammasome plays a critical role in the innate immune system against invading pathogens as the AIM2 sensor is capable of detecting foreign DNA. Despite extensive studies on the operating mode of the AIM2-ASC inflammasome, the interplay between AIM2, ASC and DNA in inflammasome formation is not fully understood. Particularly, the kinetic parameters involved in AIM2-ASC inflammasome assembly are not known and a knowledge gap exists regarding the molecular bases of AIM2 regulation. Filling these gaps will facilitate identifying molecular interventions for the manipulation of inflammasomes. Single-molecule techniques require sub-nanomolar protein concentrations thus enabling kinetic studies on inflammasome assembly before massive oligomerization occurs. In particular, optical tweezers can leverage AIM2-DNA binding and allow full mechanical control of the single DNA molecule. By combining optical tweezers, confocal fluorescence microscopy for real- time visualization and microfluidics for stepwise addition of inflammasome components, this proposal aims at forming and visualizing in a cell-free system and in real-time the AIM2-ASC inflammasome. Evidence is presented on the application of this strategy showing real-time assembly of AIM2 oligomers stochastically bound to a single DNA molecule tethered by two optically trapped beads. These promising results will be capitalized to determine the concerted roles of full-length ASC and AIM2 in inflammasome assembly (aim 1) and to establish the mechanism of action of a natural inhibitor of AIM2 on inflammasome regulation (aim 2). The overarching significance of the project is to obtain information on the mechanisms of inflammasome activation and regulation by overcoming current limitations using the proposed strategy: a) providing unknown information on the kinetics and assembly of the AIM2-ASC inflammasome; b) setting the grounds for the extension to other inflammasomes to obtain comprehensive relational data on inflammasome operating modes; c) testing inflammasome inhibitors to gain in-depth knowledge on their function at the molecular level; d) facilitating the design of molecular models of disease resulting from excessive inflammasome activation.