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

项目总结/摘要。炎症是先天免疫系统的主要反应， 感染它的失调导致慢性炎症，这是危及生命的疾病的主要原因。发作 炎症的发生依赖于称为炎性小体的多蛋白复合物的细胞内组装， 其在有害物质存在时通过三种蛋白质的多个拷贝的寡聚化形成： 对来自病原体或受损组织的危险信号作出反应的传感器; -激活 - 适配器ASC，其通过连接传感器和 半胱天冬酶原-1分子。研究控制炎性小体组装的分子机制是 具有挑战性，因为它的动态性质和炎性小体相关蛋白自我调节的强烈倾向， 合伙人这些挑战限制了我们对炎性小体组装的理解。AIM2-ASC 炎性小体作为AIM 2传感器在先天免疫系统中对抗入侵病原体起着关键作用 能够检测到外来DNA尽管对AIM 2-ASC的操作模式进行了广泛的研究， 尽管AIM 2和ASC与炎性小体之间存在相互作用，但AIM 2、ASC和DNA在炎性小体形成中的相互作用尚未完全了解。 特别地，涉及AIM 2-ASC炎性小体组装的动力学参数是未知的， 关于AIM 2调节的分子基础存在知识缺口。填补这些空白将有助于 鉴定用于操纵炎性小体的分子干预。单分子技术需要 亚纳摩尔蛋白浓度，从而使得能够在大规模前对炎性小体组装进行动力学研究。 发生低聚。特别地，光镊可以利用AIM 2-DNA结合，并允许完全机械的结合。 控制单个DNA分子。通过结合光镊，共聚焦荧光显微镜的真实的- 时间可视化和微流体用于逐步添加炎性体组分，该提议旨在 在无细胞系统中形成并实时显示AIM 2-ASC炎性小体。证据 介绍了应用这种策略显示实时组装AIM 2寡聚体随机结合 一个DNA分子被两个光学捕获的珠子所束缚。这些有希望的结果将被资本化， 确定全长ASC和AIM 2在炎性小体组装中的协同作用（目的1），并建立 AIM 2天然抑制剂对炎性小体调节的作用机制（目的2）。总体 该项目的意义在于获得炎性小体激活和调节机制的信息 通过使用所提出的策略克服当前的限制：a）提供关于动力学的未知信息 和AIM 2-ASC炎性小体的组装; B）为延伸至其它炎性小体奠定基础 以获得关于炎性体操作模式的综合相关数据; c）测试炎性体抑制剂 深入了解它们在分子水平上的功能; d）促进分子模型的设计 炎症体过度激活导致的疾病。

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.