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

细胞质中的DNA识别激活干扰素的产生。DDX 41是一种死亡盒蛋白，已被鉴定为DNA传感器之一。STING是一种膜结合蛋白，其充当DNA传感器和干扰素应答激活之间的介体。这两种蛋白质也参与激活细胞先天免疫反应，以响应宿主或细菌来源的环状二核苷酸。到目前为止，尚不清楚DDX 41和STING的激活和相互作用如何在分子水平上发挥作用。在这里，我们计划在体外表征DDX 41的DNA和环状二核苷酸识别，并确定配体结合后蛋白质的构象变化。此外，我们将使用生物化学，生物物理和单分子荧光技术的组合来了解STING激活中涉及的分子事件。特别地，我们将I）研究STING的配体诱导的二聚化/寡聚化，II）跟踪STING胞质结构域和C末端尾在环状二核苷酸结合后的构象变化，以及III）研究STING-DDX 41相互作用以理解STING如何通过传感器结合激活。

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.