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

Toll样受体核酸识别的结构基础

• 批准号: 8704350
• 负责人: Yorgo Modis
• 金额: $ 10.58万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8704350
• 关键词: Affinity; Anti-Inflammatory Agents; Anti-inflammatory; Apoptosis; Asthma; Atomic Resolution X-Ray Crystallography; Autoantigens; Autoimmune Diseases; Bacteria; Bacterial DNA; Base Pairing; Binding; Biochemical; C-terminal; Cells; Chemicals; Chemistry; Cleaved cell; Complex; Cryoelectron Microscopy; Cytoplasm; DNA; DNA Binding; DNA Structure; Data; Detergents; Dimerization; Endosomes; Event; Extracellular Structure; Family; Genetic Materials; Graft Rejection; HMGB1 gene; Higher Order Chromatin Structure; Histone H2A; Histones; Immune; Immunologic Receptors; In Vitro; Infection; Inflammatory Response; Injury; Invaded; Length; Life; Ligand Binding; Ligands; Liposomes; Lupus; Measures; Methods; Methylation; Micelles; Molecular; Mutation; N-terminal; Nucleic Acids; Nucleosomes; Organ Transplantation; Pattern; Peptide Hydrolases; Post-Translational Protein Processing; Property; Proteins; RNA; Recombinants; Relative (related person); Septic Shock; Signal Transduction; Site; Structure; Syndrome; Systemic Lupus Erythematosus; TLR3 gene; TLR7 gene; TLR8 gene; Testing; Therapeutic; Tissues; Toll-like receptors; Vaccine Adjuvant; Vertebral column; Virus Diseases; X-Ray Crystallography; base; biophysical techniques; fighting; human TLR7 protein; image reconstruction; immunogenic; in vivo; insight; microbial; novel; nucleic acid localization; particle; pathogen; phosphodiester; phosphorothioate; protein complex; receptor; reconstitution; reconstruction; response; tool; viral RNA

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.

描述（由申请人提供）：识别广泛保守的微生物细胞外结构时，Toll样受体（TLR）会产生先天免疫信号反应。病毒RNA被！TLR3，TLR7和TLR8识别，而微生物DNA识别为TLR9。直到最近，现行的范式是TLR9识别未甲基化的CpG DNA基序，这些基序在细菌中很丰富，但在哺乳动物DNA中相对较少。然而，最近的研究在内，包括我们自己的初步数据表明，TLR9独立于其序列和甲基化状态结合天然DNA配体。我们提出了对允许TLR9识别微生物DNA的结构特性的全面分析，包括序列，长度，双工含量，甲基化状态，骨链化学（磷酸二酯与磷酸硫酸磷酸盐），曲率和高阶结构（例如连接）。我们在初步研究中表明，DNA曲率诱导蛋白！HMGB1和组蛋白H2A和H2B显着增强了与TLR9的C末端裂解片段的结合，这表明TLR9优先识别弯曲的DNA骨架。为了确定单独的DNA曲率负责结合增强的程度，我们建议测量含有75至120个碱基对的DNA微圆的TLR9结合亲和力。由于核小体可以诱导TLR依赖性自身免疫原性信号传导，因此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！这些体外研究将在体内验证，通过测量用各种DNA或蛋白-DNA配体刺激的细胞中TLR9依赖的信号反应，包括微量圆形，核小体，连接体和甲基化的DNA配体。必须将TLR7/8/9的外域裂解以产生能够信号传导的受体。在裂解TLR9的第一项研究中，我们在初步数据中表明，N-和C末端TLR9胞外域片段都参与配体结合和受体二聚体。因此，我们假设这两个片段在内体中的蛋白水解裂解后保持相关性，并且TLR9可能需要裂解，即可经历激活受体的配体诱导的构象变化。为了检验这一假设，我们将探讨两个TLR9胞外域片段之间的物理和功能关系，并使用生物物理方法阐明蛋白水解活化的物理基础。缺乏TLR7/8/9的结构信息限制了我们对这些受体识别核酸识别的理解。我们建议将电子冷冻显微镜和X射线晶体学用作互补方法，以深入了解TLR9-DNA识别的结构基础。通过提供TLR9对微生物DNA识别的分子水平，该项目将提供必要的工具来创建更有效的疫苗佐剂，并提供一类新的抗炎疗法，并具有广泛的应用，包括特定系统性的Erythematosus themthmatosus，atthma shatic，spticect，spictic震撼，造型器官和机器人的转换剂。