Functional characterization of sensor DDX41 and mediator STING involved in the innate immune response to cytosolic DNA and cyclic dinucleotides

胞浆中的DNA识别激活了干扰素的产生。DDX41是一种死盒蛋白，已被鉴定为DNA传感器之一。SING是一种膜结合蛋白，在DNA传感器和干扰素反应的激活之间发挥中介作用。这两种蛋白质还参与激活细胞对来自宿主或细菌来源的环二核苷酸的天然免疫反应。到目前为止，还不清楚DDX41和STING在分子水平上是如何激活和相互作用的。在这里，我们计划在体外研究DDX41在DNA和环二核苷酸识别方面的特征，并确定蛋白质在配体结合时发生的构象变化。此外，我们将使用生化、生物物理和单分子荧光技术相结合的方法来了解与刺痛激活有关的分子事件。特别是，我们将研究配体诱导的STIN二聚/寡聚，II)跟踪STIN胞液域和C末端尾部在环二核苷酸结合时的构象变化，以及III)研究STINDDX41相互作用以了解传感器结合是如何激活STING的。

DNA recognition in the cytosol activates interferon production. DDX41 is a DEAD-box protein that has been identified as one of the DNA sensors. STING is a membrane bound protein that acts as a mediator between the DNA sensors and the activation of interferon response. These two proteins have also been involved in activating cell innate immune reaction in response to cyclic dinucleotides, either of host or bacterial origin. So far it is unclear how DDX41 and STING activation and interaction function on a molecular level. Here, we plan to characterize DDX41 in vitro with respect to DNA and cyclic dinucleotide recognition and identify the conformational changes the protein undergoes upon ligand binding. In addition, we will use a combination of biochemical, biophysical and single molecule fluorescence techniques to understand the molecular events involved in STING activation. In particular we will I) investigate ligand-induced dimerization/oligomerization of STING, II) follow conformational changes of STING cytosolic domain and C-terminal tail upon cyclic dinucleotide binding, and III) study STING-DDX41 interaction to understand how STING is activated by sensor binding.