天然免疫应答中DNA结合蛋白DAI（ZBP1/DLM1）的结构与功能研究

In vertebrate, innate immunity plays important roles in defending the infection of viruses and bacteria. DNA-dependent activator of IFN-regulatory factor (DAI, also called ZBP1/DLM1) is a cytosolic DNA sensor for detecting DNA from viruses and bacteria. DAI induces the activation of multiple signaling pathways including NF-κB activation and type I interferon (IFN) expression. The single domain structure (Zα and Zβ) was solved by X-ray crystallography. However, it is not clear how Zαβ domain of DAI binds with DNA synergically, activating the downstream signals, and therefore inducing the innate immunity response. Our preliminary data showed that the RHIM of DAI forms β amyloid structure in vitro. We propose RHIM of DAI forms filamentous structure after Zαβ binding with DNA, acting as a platform to amplify the downstream signals including NF-κB activation and type I IFN expression. In this proposal, the combination of structural biology (X-ray crystallography and EM), biochemical and cell biology methods is used to study the structural basis of DAI/DNA complex, and the oligomerization of RHIM. In addition, we will study the molecular mechanism of the interaction between DAI and its partners RIP1, RIP3, and TBK1 that induces NF-κB and type I IFN signals. It would greatly benefit our understanding for the molecular mechanism of sensing pathogen DNA in innate immunity.

天然免疫在脊椎动物抵抗病毒和细菌感染中起着重要作用。DNA结合蛋白DAI（又称ZBP1/DLM1）感知病毒和细菌DNA，激活I型干扰素（IFN）的表达和NF-κB信号转导。目前已解析出DAI中单个DNA结合区域（Zα和Zβ）的晶体结构，但其完整DNA结合区（Zαβ）如何协调结合DNA，放大应答信号，从而激活天然免疫应答的分子机制仍然不清楚。我们推测Zαβ结构域结合DNA，促进DAI中RHIM结构域高聚化，形成一个级联放大平台以激活下游的NF-κB和IFN信号。本课题设想结合结构生物学（X光晶体衍射和电子显微镜）、生物化学和细胞生物学手段，获得DAI/DNA复合物的三维精细结构，阐述RHIM结构域高聚的分子基础，以及DAI与下游蛋白RIP1、RIP3和TBK1相互作用激活NF-κB和IFN信号的分子机理。本研究的成功实施将加深我们对抗病毒天然免疫系统中，感知DNA信号作用机制的理解。

天然免疫在脊椎动物抵抗病毒和细菌感染中起着重要作用。DNA结合蛋白DAI（也叫ZBP1/DLM1）感知病毒和细菌DNA，激活I型干扰素（IFN）产生和NF-κB信号转导，但其具体作用的分子机制仍不清楚。本项目通过荧光偏振实验发现DAI特异性结合B-DNA，解析了15bp长度Z-DNA的晶体结构，初步获得了DAI/DNA复合物的三维结构；发现DAI中的RHIM结构域高聚成淀粉样纤维，并通过X-光晶体衍射方法解析了IQIG高分辨率结构；相关成果将进一步加深我们对抗病毒天然免疫系统中，感知DNA信号作用机制的理解。此外，基于本项目我们拓展了包含RHIM结构域蛋白IMD、PGRP-LC和PGRP-LE的结构和功能研究，发现RHIM结构域可以跨物种发挥作用，在免疫信号应答和细胞死亡中起着重要作用。本项目共在Immunity、PNAS等著名期刊发表基金标注SCI通讯作者论文7篇。