Mechanistic Studies of cytosolic double-stranded DNA sensing pathways.

项目总结 在后生动物中，胞质中存在双链(DS)dna标志着严重的问题，范围 从辐射损伤到病原体入侵。先天免疫系统是第一道防线。 通过启动炎症信号通路对抗胞浆dsDNA。胞质dsDNA传感通路有 宿主对多种病原体的防御是不可或缺的，它们的故障也会导致各种人类 疾病。 我们的研究计划准备解决几个长期存在的机械问题，以了解如何 胞质dsDNA传感通路在分子水平上被激活和调节。我们还将测试新的 关于这些传感器如何被污名化为自身抗原的概念，并探索机械力 来驱动和调节它们更高级别的组件。我们的方法包括X射线结晶学，严谨 生化测量、基于细胞的分析、电子显微镜和单分子方法。 在这里，我们将确定cGAS如何在不同的核苷酸底物和金属辅助因子 活性部位专门产生2‘-5’/3‘-5’连接的环状G/AMP。我们还将调查二聚化是如何 变构偶联到活化。然后我们将确定cGAS和ALR传感器如何选择性地识别 并通过dsDNA发出信号。接下来，我们将测试相分离/转变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。 这为胞质dsDNA传感器的激活和调控奠定了基础。分子洞察力来自于 拟议的研究将为开发针对各种不同疾病的新治疗策略提供基础。 由胞浆dsdna传感通路失调引起的人类疾病。

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.