FRET based imaging of cyclic dinucleotide dynamics in living systems

基于 FRET 的生命系统中环状二核苷酸动力学成像

• 批准号: 10183159
• 负责人: Joshua Woodward
• 金额: $ 20.72万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10183159
• 关键词: Address; Autoimmune Diseases; Bacteria; Bacterial DNA; Bacterial Infections; Binding; Biochemical; Biological; Biological Assay; Biosensor; C-terminal; Cells; Chlamydia trachomatis; Communities; Cytoplasm; DNA; DNA Damage; Detection; Development; Dinucleoside Phosphates; Disease; Enzyme-Linked Immunosorbent Assay; Eukaryota; Eukaryotic Cell; Fluorescence Resonance Energy Transfer; Frequencies; Gap Junctions; Genetic Screening; Heterogeneity; Human; Hybrids; Image; Immune response; Immunity; Immunomodulators; In Vitro; Infection; Inflammation; Inflammation Mediators; Inflammatory; Inflammatory Response; Intervention; Listeria monocytogenes; Lupus; Malignant - descriptor; Measures; Mediating; Membrane Transport Proteins; Metabolism; Methods; Modeling; Molecular; Monitor; Movement; Mycobacterium tuberculosis; Nucleotides; Organism; Periodicity; Population; Post-Translational Regulation; Process; Production; Prokaryotic Cells; Purines; Pyrimidine; Recombinants; Regulation; Reporter; Reporting; Research Personnel; Resources; Role; SLC19A1 gene; Second Messenger Systems; Shapes; Signal Pathway; Signal Transduction; Signaling Molecule; Staphylococcus aureus; Technology; Validation; Viral; Virus Diseases; Work; base; cell growth regulation; enzyme activity; extracellular; genome-wide; human disease; in vivo; innovation; insight; new technology; novel; phosphoric diester hydrolase; pseudotoxoplasmosis syndrome; receptor; response; sensor; small molecule; technology development; temporal measurement; tissue culture; tool; viral DNA; viral detection

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.

项目摘要 宿主和细菌起源的环状二核苷酸已成为无处不在的第二使者 以及宿主免疫反应的有效调节剂，在塑造感染性中具有重要作用， 恶性和自身免疫性疾病。产生真核生物第二信使2'，3'-cgamp 通过CGA响应宿主细胞细胞质内的DNA。响应于源自的DNA 细菌和病毒感染CGAMP引起宿主炎症以清除感染，同时感应 自源性DNA的自身免疫性疾病（包括全身性狼疮）与 红细胞和AICARDI-GOTIERES综合征。另外，细菌产生多种环状 作为第二使者的二核苷酸，还会促进宿主炎症 感染。在每种情况下，CDN与哺乳动物受体刺的结合都会促进 炎症反应。尽管我们目前与CDN有关的理解 炎症，直接测量和观察的能力有很大的限制 生物环境中的CDN。迄今为止，CDN检测依赖于LC-MS/MS或基于ELISA的检测 方法。这些技术虽然重要的空间和时间分辨率受到限制 他们负担得起。为了克服这些目前的局限，我们进行了发展，并 验证通用，遗传编码的荧光CDN生物传感器。这个传感器依靠 刺激的CDN结合结构域，并提供了无与伦比的时间和单细胞检测 活细胞中的CDN。现在，我们的目标是（i）生化表征并建立体外实用程序 该传感器用于监测CDN动力学，（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，（iii）将此新技术应用于 对人的CGAS-CGAMP信号的细胞内在调节剂进行正向遗传筛选 细胞。此处概述的研究将提供一种创新且广泛有用的工具 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信号传导轴，对感染性，恶性和 自身免疫性疾病。