The structural basis of nucleic acid recognition by Toll-like receptors

描述(申请人提供)：Toll样受体(TLRs)在识别广泛保守的微生物细胞外结构时产生先天免疫信号反应。TLR3、TLR7和TLR8识别病毒RNA，TLR9识别微生物DNA。直到最近，流行的范式是TLR9识别未甲基化的CpG DNA基序，这些基序在细菌中丰富，但在哺乳动物DNA中相对稀少。然而，包括我们自己的初步数据在内的最近的研究表明，TLR9与自然DNA配体的结合不依赖于它们的序列和甲基化状态。我们建议对使TLR9能够识别微生物DNA的结构特性进行综合分析，包括序列、长度、双链含量、甲基化状态、骨架化学(磷酸二酯与硫代磷)、曲率和高阶结构(如连接)。我们在初步研究中发现，DNA曲率诱导蛋白！HMGB1以及组蛋白H_2A和H_2B显著增强与TLR9的C端切割片段的结合，表明TLR9优先识别弯曲的DNA骨架。为了确定DNA曲率本身对结合增强的影响程度，我们建议测量包含75到120个碱基对的DNA小环的TLR9结合亲和力。由于核小体可以诱导TLR依赖的自身免疫原信号，我们建议将整个核小体作为TLR9配体进行体外生物物理分析！这些体外研究将通过测量细胞中依赖TLR9的信号反应来验证，这些细胞受到各种DNA或蛋白质-DNA配体的刺激，包括微环、核小体、连接和甲基化DNA配体。TLR7/8/9胞外区必须被蛋白水解性切割，才能产生能够传递信号的受体。在第一次对切割的TLR9的研究中，我们在我们的初步数据中表明，N端和C端的TLR9胞外结构域片段都参与了配体结合和受体二聚。因此，我们假设，这两个片段在内体中被蛋白质降解切割后仍然是相关的，并且切割可能是TLR9经历配体诱导的构象变化以激活受体所必需的。为了验证这一假说，我们将探索两个TLR9胞外结构域片段之间的物理和功能关系，并利用生物物理方法阐明蛋白水解酶激活的物理基础。缺乏TLR7/8/9的结构信息限制了我们对这些受体识别核酸的理解。我们建议使用电子冷冻显微镜和X射线结晶学作为互补的方法，以深入了解TLR9-DNA识别的结构基础。通过提供对TLR9识别微生物DNA的分子水平的了解，该项目将提供必要的工具来创造更有效的疫苗佐剂，以及具有广泛应用的新型抗炎疗法，特别是包括系统性红斑狼疮、哮喘、败血症休克综合征和器官移植排斥反应。

DESCRIPTION (provided by applicant): Toll-like receptors (TLRs) generate an innate immune signaling response upon recognizing broadly conserved microbial extracellular structures. Viral RNA is recognized by!TLR3, TLR7 and TLR8, and microbial DNA is recognized by TLR9. Until recently the prevailing paradigm was that TLR9 recognized unmethylated CpG DNA motifs, which are abundant in bacteria but relatively scarce in mammalian DNA. However, recent studies including our own preliminary data suggest that TLR9 binds natural DNA ligands independently of their sequence and methylation state. We propose a comprehensive analysis of the structural properties that allow TLR9 to recognize microbial DNA including sequence, length, duplex content, methylation state, backbone chemistry (phosphodiester versus phosphorothioate), curvature and higher order structure (such as junctions). We show in preliminary studies that DNA curvature-inducing proteins!HMGB1 and histones H2A and H2B significantly enhance binding to the C-terminal cleavage fragment of TLR9, suggesting that TLR9 preferentially recognizes curved DNA backbones. To determine the extent to which DNA curvature alone is responsible for the binding enhancement, we propose to measure the TLR9 binding affinity of DNA minicircles containing 75 to 120 base pairs. Since nucleosomes can induce TLR-dependent auto immunogenic signaling, we propose an in vitro biophysical analysis of whole nucleosomes as TLR9 ligands! These in vitro studies will be validated in vivo by measuring TLR9-dependent signaling responses in cells stimulated with various DNA or protein-DNA ligands including minicircles, nucleosomes, junctions and methylated DNA ligands. The TLR7/8/9 ectodomains must be proteolytically cleaved in order to produce receptors that are capable of signaling. In the first study with cleaved TLR9, we show in our preliminary data that both the N- and C-terminal TLR9 ectodomain fragments participate in ligand binding and receptor dimerization. We therefore hypothesize that the two fragments remain associated after proteolytic cleavage in the endosome, and that cleavage may be necessary for TLR9 to undergo the ligand-induced conformational change that activates the receptor. To test this hypothesis, we will explore the physical and functional relationships between the two TLR9 ectodomain fragments, and elucidate the physical basis of proteolytic activation using biophysical approaches. The lack of structural information for TLR7/8/9 limits our understanding of nucleic acid recognition by these receptors. We propose to use electron cryomicroscopy and X-ray crystallography as complementary approaches to gain insight into the structural basis of TLR9-DNA recognition. By providing a molecular-level understanding of the recognition of microbial DNA by TLR9, this project will provide the necessary tools to create more potent vaccine adjuvants, and a new class of anti-inflammatory therapeutics with a wide range of applications including in particular systemic lupus erythematosus, asthma, septic shock syndrome and organ transplant rejection.