FRET based imaging of cyclic dinucleotide dynamics in living systems

项目总结 宿主和细菌来源的环二核苷酸已成为普遍存在的第二信使 和宿主免疫反应的有效调节剂，在形成传染性， 恶性和自身免疫性疾病。真核细胞第二信使2‘，3’-cGAMP的产生 通过cGAS对宿主细胞胞质内的DNA作出反应。作为对来自于 细菌和病毒感染cGAMP启动宿主炎症以清除感染，同时感知 与包括系统性红斑狼疮在内的自身免疫性疾病有关 红细胞症和Aicardi-Goutieres综合征此外，细菌还会产生各种环状物质 二核苷酸起第二信使的作用，也促进宿主在 感染。在每一种情况下，CDN与哺乳动物受体刺痛结合促进 炎症反应。尽管我们目前对CDN介导的 炎症，直接测量和观察的能力有很大限制 生物环境中的CDN。到目前为止，CDN的检测依赖于LC-MS/MS或基于ELISA法 方法：研究方法。这些技术虽然重要，但在空间和时间分辨率上是有限的 他们负担得起。为了克服这些目前的限制，我们已经进行了开发和 一种通用的、基因编码的CDN荧光生物传感器的验证。这个传感器依赖于 STING的CDN结合域提供了无与伦比的时间和单细胞检测 活细胞中的CDN。我们现在的目标是(I)生物化学特征和建立体外实用 用于监测CDN动态的传感器，(Ii)利用组织培养研究来验证和 描述活细胞中CDN水平的动态，以及(Iii)将这项新技术应用于 对人类cGAS-cGAMP信号的细胞内在调节因子进行正向遗传筛选 细胞。总之，这里概述的研究将提供一个创新的和广泛有用的工具，以 研究真核生物中的CDN信号并为其调控提供潜在的生物学见解 CGAS-cGAMP信号轴，对感染性、恶性和 自身免疫性疾病。

PROJECT SUMMARY Cyclic dinucleotides of host and bacterial origin have emerged as ubiquitous second messengers and potent modulators of host immune responses, with important roles in shaping infectious, malignant and autoimmune diseases. The eukaryotic second messenger 2',3'-cGAMP is produced by cGAS in response to DNA within the host cell cytoplasm. In response to DNA derived from bacterial and viral infection cGAMP initiates host inflammation to clear infection, while sensing of self-derived DNA has been implicated in autoimmune disorders including Systemic Lupus Erythemytosus and Aicardi-Goutieres Syndrom. Additionally, bacteria produce a variety of cyclic dinucleotides that function as second messengers and also promote host inflammation during infection. In each of these instances, CDN binding to the mammalian receptor STING promotes inflammatory responses. Despite our current understanding pertaining to CDN mediated inflammation, there is a significant limitation in the capacity to directly measure and observe CDNs within biological settings. To date, CDN detection relies on LC-MS/MS or ELISA based methods. These technologies while important are limited in the spatial and temporal resolution they afford. To overcome these current limitations, we have undertaken the development and validation of a universal, genetically encoded fluorescent CDN biosensor. This sensor relies on the CDN binding domain of STING and affords unparalleled temporal and single cell detection of CDNs in living cells. We now aim to (i) biochemically characterize and establish the in vitro utility of this sensor for monitoring CDN dynamics, (ii) utilize tissue culture studies to validate and characterize the dynamics of CDN levels in living cells, and (iii) apply this new technology to conduct a forward genetic screen for cell intrinsic regulators of cGAS-cGAMP signaling in human cells. Together the studies outlined here will provide an innovative and broadly useful tool to study CDN signaling within eukaryotes and provide potential biological insight into the regulation of the cGAS-cGAMP signaling axis, with important consequences on infectious, malignant, and autoimmune diseases.