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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要宿主和细菌来源的环状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。产生真核第二信使 2',3'-cGAMP cGAS 响应宿主细胞胞质内的 DNA。响应源自 DNA 细菌和病毒感染 cGAMP 启动宿主炎症以清除感染，同时感知自体衍生 DNA 与包括系统性狼疮在内的自身免疫性疾病有关红斑狼疮和 Aicardi-Goutieres 综合征。此外，细菌还产生多种循环作为第二信使的二核苷酸也可促进宿主炎症感染。在每种情况下，CDN 与哺乳动物受体 STING 的结合都会促进炎症反应。尽管我们目前对 CDN 介导的了解炎症，直接测量和观察的能力存在很大限制生物环境中的 CDN。迄今为止，CDN 检测依赖于基于 LC-MS/MS 或 ELISA 的方法。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。为了克服当前的这些限制，我们进行了开发和通用基因编码荧光 CDN 生物传感器的验证。该传感器依赖于 STING 的 CDN 结合域，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) 进行生化表征并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) 将这项新技术应用于对人类 cGAS-cGAMP 信号传导的细胞内在调节因子进行正向遗传筛选细胞。这里概述的研究将共同提供一个创新且广泛有用的工具研究真核生物内的 CDN 信号传导并提供对调控的潜在生物学见解 cGAS-cGAMP 信号轴的作用，对传染性、恶性和自身免疫性疾病。