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）：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免疫信号反应。病毒RNA被TLR3、TLR7和TLR8识别，微生物DNA被TLR9识别。直到最近，流行的模式是 TLR9 识别未甲基化的 CpG DNA 基序，这种基序在细菌中大量存在，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，TLR9 与天然 DNA 配体结合，与其序列和甲基化状态无关。我们建议对 TLR9 识别微生物 DNA 的结构特性进行全面分析，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）、曲率和高阶结构（例如连接）。我们在初步研究中表明，DNA 弯曲诱导蛋白！HMGB1 以及组蛋白 H2A 和 H2B 显着增强与 TLR9 C 末端切割片段的结合，表明 TLR9 优先识别弯曲的 DNA 主链。为了确定 DNA 曲率单独对结合增强负责的程度，我们建议测量包含 75 至 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 依赖性自身免疫原性信号传导，因此我们建议对整个核小体作为 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 TLR9 依赖性信号传导反应来进行体内验证。 TLR7/8/9 胞外域必须经过蛋白水解裂解才能产生能够发出信号的受体。在第一项针对切割 TLR9 的研究中，我们在初步数据中显示 N 端和 C 端 TLR9 胞外域片段均参与配体结合和受体二聚化。因此，我们假设在内体中蛋白水解裂解后这两个片段仍然相关，并且裂解可能是 TLR9 经历配体诱导的构象变化（激活受体）所必需的。为了检验这一假设，我们将探索两个 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激活的物理基础。 TLR7/8/9 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。我们建议使用电子冷冻显微镜和 X 射线晶体学作为补充方法来深入了解 TLR9-DNA 识别的结构基础。通过在分子水平上了解 TLR9 对微生物 DNA 的识别，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治疗药物，特别是系统性红斑狼疮、哮喘、败血性休克综合征和器官移植排斥反应。