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

描述（由申请人提供）：Toll样受体（TLR）在识别广泛保守的微生物细胞外结构后产生先天性免疫信号传导应答。病毒RNA是公认的！TLR 3、TLR 7和TLR 8，微生物DNA由TLR 9识别。直到最近，流行的范例是TLR 9识别未甲基化的CpG DNA基序，其在细菌中丰富，但在哺乳动物DNA中相对稀少。然而，最近的研究，包括我们自己的初步数据表明，TLR 9结合天然DNA配体独立于它们的序列和甲基化状态。我们提出了一个全面的分析的结构特性，允许TLR 9识别微生物DNA，包括序列，长度，双链体含量，甲基化状态，骨架化学（磷酸二酯与硫代磷酸酯），曲率和更高的顺序结构（如路口）。我们在初步研究中表明，DNA弯曲诱导蛋白质！HMGB 1和组蛋白H2 A和H2 B显著增强与TLR 9的C-末端切割片段的结合，表明TLR 9优先识别弯曲的DNA骨架。为了确定在何种程度上，DNA曲率单独负责的结合增强，我们建议测量TLR 9的结合亲和力的DNA小环含有75至120个碱基对。由于核小体可以诱导TLR依赖的自身免疫原性信号，我们提出了一个体外生物物理分析的整个核小体作为TLR 9配体！这些体外研究将通过测量用各种DNA或蛋白质-DNA配体（包括微环、核小体、连接和甲基化DNA配体）刺激的细胞中的TLR 9依赖性信号传导应答来在体内验证。TLR 7/8/9胞外域必须被蛋白水解切割以产生能够信号传导的受体。在第一项研究中，裂解的TLR 9，我们在我们的初步数据表明，N-和C-末端TLR 9胞外域片段参与配体结合和受体二聚化。因此，我们假设这两个片段在内体中蛋白水解切割后保持相关，并且切割可能是TLR 9进行激活受体的配体诱导的构象变化所必需的。为了验证这一假设，我们将探讨两个TLR 9胞外域片段之间的物理和功能关系，并阐明蛋白水解激活的物理基础，使用生物物理方法。TLR 7/8/9结构信息的缺乏限制了我们对这些受体的核酸识别的理解。我们建议使用电子低温显微镜和X射线晶体学作为互补的方法来深入了解TLR 9-DNA识别的结构基础。通过提供TLR 9识别微生物DNA的分子水平的理解，该项目将提供必要的工具来创建更有效的疫苗佐剂，以及一类新的抗炎治疗药物，其应用范围广泛，特别是系统性红斑狼疮，哮喘，脓毒性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.