FRET based imaging of cyclic dinucleotide dynamics in living systems

项目摘要 宿主和细菌来源的环状二核苷酸已经成为普遍存在的第二信使 和宿主免疫反应的有效调节剂，在形成感染性， 恶性肿瘤和自身免疫性疾病。产生真核第二信使2 '，3'-cGAMP 通过CGAS响应宿主细胞质内的DNA。DNA来源于 细菌和病毒感染cGAMP引发宿主炎症以清除感染，同时感测 自身衍生的DNA与包括系统性狼疮在内的自身免疫性疾病有关 红细胞增多症和Aicardi-Goutieres综合征此外，细菌还产生各种循环 二核苷酸作为第二信使，也促进宿主炎症， 感染在这些情况中的每一种中，CDN与哺乳动物受体STING的结合促进了哺乳动物细胞的增殖。 炎症反应。尽管我们目前的理解有关CDN介导的 炎症，直接测量和观察的能力有很大的局限性， 生物环境中的CDN。迄今为止，CDN检测依赖于基于LC-MS/MS或ELISA的方法。 方法.这些技术虽然重要，但在空间和时间分辨率方面受到限制 他们负担得起。为了克服目前的这些局限性，我们已经着手开发和 通用的遗传编码的荧光CDN生物传感器的验证。这个传感器依赖于 STING的CDN结合结构域，并提供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）生物化学表征和建立体外效用 该传感器用于监测CDN动态，（ii）利用组织培养研究来验证和 表征活细胞中CDN水平的动态，以及（iii）将这种新技术应用于 在人类中进行cGAS-cGAMP信号传导的细胞内在调节剂的正向遗传筛选 细胞这里概述的研究将提供一个创新和广泛有用的工具， 研究真核生物中的CDN信号传导，并为调节提供潜在的生物学见解 cGAS-cGAMP信号轴，对感染性，恶性， 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.