Mechanistic Studies of cytosolic double-stranded DNA sensing pathways.

胞质双链 DNA 传感途径的机制研究。

• 批准号: 10689702
• 负责人: JUNGSAN SOHN
• 金额: $ 40.94万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10689702
• 关键词: Active Sites; Autoantigens; Biochemical; Biological Assay; Cells; Coupled; Cytosol; Dimerization; Disease; Electron Microscopy; Foundations; Host Defense; Human; Inflammatory; Innate Immune System; Invaded; Link; Measurement; Metals; Methods; Molecular; Nucleotides; Pathway interactions; Periodicity; Phase; Radiation induced damage; Regulation; Research; Role; Signal Pathway; Signal Transduction; Stigmatization; Testing; X-Ray Crystallography; cofactor; ds-DNA; human disease; insight; mechanical force; novel therapeutic intervention; pathogen; programs; public health relevance; sensor; single molecule; targeted treatment; therapy development

Project summary In metazoan, the presence of double-stranded (ds)DNA in the cytosol signals serious problems, which range from radiation damage to pathogen invasion. The innate immune system acts as the first line of defense against cytosolic dsDNA by initiating inflammatory signaling pathways. Cytosolic dsDNA sensing pathways are integral to host defense against numerous pathogens, and their malfunctions also result in various human maladies. Our research program is poised to resolve several long-standing mechanistic questions in understanding how cytosolic dsDNA sensing pathways are activated and regulated at the molecular level. We will also test new concepts as to how these sensors might be stigmatized as autoantigens, and explore the mechanical forces that drive and regulate their higher order assemblies. Our approaches include X-ray crystallography, rigorous biochemical measurements, cell-based assays, electron microscopy, and single molecule methods. Here, we will determine how cGAS coordinates different nucleotide substrates and metal co-factors at the active site to specifically generate 2'-5'/3'-5' linked cyclic G/AMP. We will also investigate how dimerization is allosterically coupled to activation. We will then determine how cGAS and ALR sensors selectively recognize and signal through dsDNA. Next, we will test the role of phase-separation/transition in the normal and aberrant activities of cytosolic dsDNA sensors. Finally, we will delineate mechanical forces and structural mechanisms that underpin the activation and regulation of cytosolic dsDNA sensors. The molecular insights resulting from the proposed studies will provide a foundation for developing new therapeutic strategies that target various human diseases caused by dysregulated cytosolic dsDNA sensing pathways.

项目摘要 在多田族中，在胞质溶胶中存在双链（DS）DNA，这是严重问题的严重问题 从辐射损害到病原体入侵。先天免疫系统是第一道防线 通过启动炎症信号通路来抵抗胞质dsDNA。胞质DSDNA传感途径是 托管防御众多病原体的不可或缺的一部分，它们的故障也导致了各种人类 疾病。 我们的研究计划有望解决几个长期的机械问题，以理解如何 胞质DSDNA感应途径被激活并在分子水平上调节。我们还将测试新的 关于这些传感器如何被污名化为自动抗原的概念，并探索机械力 驱动并规范其高阶组件。我们的方法包括X射线晶体学，严格 生化测量，基于细胞的测定，电子显微镜和单分子方法。 在这里，我们将确定CGA如何在不同的核苷酸底物和金属辅助因子坐标 主动位点以特异性生成2'-5'/3'-5'链接的循环g/amp。我们还将调查二聚化的 变构与激活结合。然后，我们将确定CGA和ALR传感器如何有选择地识别 并通过dsDNA发出信号。接下来，我们将测试相位分离/过渡在正常和异常中的作用 胞质DSDNA传感器的活性。最后，我们将描述机械力和结构机制 这是胞质DSDNA传感器的激活和调节的基础。由 拟议的研究将为制定针对各种的新治疗策略提供基础 由胞质DSDNA传感途径失调引起的人类疾病。