M. tuberculosis lipoprotein-TLR2 interactions

结核分枝杆菌脂蛋白-TLR2 相互作用

• 批准号: 7329170
• 负责人: Clifford V Harding
• 金额: $ 37.89万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-12-15 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7329170
• 关键词: Acylation; Affect; Affinity; Agonist; Amino Acid Sequence; Bacillus (bacterium); Bacteria; Bacterial Infections; Binding; Biochemical; Biological Assay; CD14 Antigen; CD14 gene; CD36 gene; Cell surface; Cells; Cellular Assay; Chimeric Proteins; Chronic; Data; Deletion Mutation; Dendritic Cells; Dependence; Detection; Engineering; Enzyme-Linked Immunosorbent Assay; Extracellular Domain; Goals; Host resistance; Human; Immune system; Immunity; Immunologic Adjuvants; Infection; Knock-out; Lead; Ligands; Lipid Binding; Lipids; Lipoprotein Binding; Lipoproteins; Maps; Measures; Molecular; Mus; Mutate; Mutation; Mycobacterium tuberculosis; N-terminal; Natural Immunity; Pattern; Peptide Sequence Determination; Personal Satisfaction; Physiological; Production; Proteins; Published Comment; Publishing; Recombinant Proteins; Recombinants; Relative (related person); Research; Research Personnel; Role; Screening procedure; Sequence Deletion; Serum-Free Culture Media; Signal Transduction; Specificity; Structural Protein; Structure; Structure-Activity Relationship; Surface; System; TLR1 gene; TLR2 gene; TLR6 gene; Tertiary Protein Structure; Testing; Therapeutic Uses; Toll-Like Receptor 2; Toll-like receptors; Tuberculosis; Variant; Wild Type Mouse; base; cytokine; design; insight; loss of function; macrophage; novel; pathogen; programs; protein structure; receptor; receptor function; research study; response; synthetic peptide; tool; tuberculosis treatment

Our goal is to determine the biochemical and structural basis for binding of Mycobacterium tuberculosis (MTB) lipoproteins to Toll-like receptor (TLR)-2 and resulting agonist activity. TLR2 recognition of MTB lipoproteins initiates innate immunity and influences adaptive immunity to MTB. Despite this critical role for TLR2 in tuberculosis, the structural basis for TLR2 recognition of MTB lipoproteins remains poorly understood. In addition, TLR2 functions in recognition of other pathogenic species, yet the structural determinants of TLR2 agonist activity are largely unexplored. It is known that acyl structures of lipoproteins influence their recognition by TLR2, but the influence of protein structures on TLR2 binding is essentially unknown. We have characterized three distinct MTB lipoproteins that signal through TLR2: LpqH (19-kDa lipoprotein), LprG and LprA. These lipoproteins are all TLR2 agonists but differ in potency and apparent structural determinants of their activity. Our data indicate that both lipid and protein components of MTB lipoproteins can influence TLR2 agonist activity. We are constructing recombinant tagged lipoproteins and soluble TLR2 fusion proteins to dissect structure-function relationships in TLR2-ligand interactions relevant to these pathophysiologically important TLR2 agonists from MTB. Aim 1 will use cellular cytokine secretion readouts to study the activity of His-tagged recombinant MTB lipoproteins and their receptor dependence (use of TLR1 or TLR6 as co-receptors in heterodimers with TLR2, as well as use of accessory receptors, CD14 and CD36). Aim 2 will determine structural features of MTB lipoproteins that affect interations with TLR2, TLR1, TLR6 and accessory receptors (CD14 and CD36) by use of macrophages and dendritic cells from mice that are genetically deficient in there receptors and analyses of MTB lipoprotein variantswithout acylation and/or with deletions, truncations or mutations in the protein sequence (or use of minimal active constructs expressed as recombinant proteins or made as synthetic peptides). Aim 3 will use direct biochemical binding assays to study binding of tagged recombinant soluble TLR and lipoprotein molecules. We will measure the affinities of different MTB lipoproteins and structural variants thereof for TLR2 to further understand the structural determinants of agonist binding to TLR2. Overall we will determine the structural basis for binding of MTB lipoproteins to TLR2, including contributions of lipid and protein components. RELEVANCE: These studies will provide unique and novel insights into the mechanisms by which TLR2 recognizes MTB. TLR2 is a key immune system receptor involved in recognition of MTB. Greater understanding of its function will help reveal important mechanisms in immunity that lead to host resistance and/or evasion of immunity during chronic infection by MTB. This may help develop better treatments for tuberculosis. It may also aid in design of better immune adjuvants for a wide array of therapeutic uses.

我们的目标是确定结核分枝杆菌结合的生化和结构基础 (MTB) 脂蛋白与 Toll 样受体 (TLR)-2 结合并产生激动剂活性。 TLR2 识别 MTB 脂蛋白启动先天免疫并影响 MTB 的适应性免疫。尽管发挥着至关重要的作用 TLR2在结核病中的作用，TLR2识别MTB脂蛋白的结构基础仍然很差 明白了。此外，TLR2 具有识别其他致病物种的功能，但其结构 TLR2 激动剂活性的决定因素很大程度上尚未被探索。众所周知，脂蛋白的酰基结构 影响它们被 TLR2 识别，但蛋白质结构对 TLR2 结合的影响本质上是 未知。我们已经表征了通过 TLR2 发出信号的三种不同的 MTB 脂蛋白： LpqH (19-kDa 脂蛋白）、LprG 和 LprA。这些脂蛋白都是 TLR2 激动剂，但效力和表观不同 其活动的结构决定因素。我们的数据表明 MTB 的脂质和蛋白质成分 脂蛋白可以影响 TLR2 激动剂活性。我们正在构建重组标记脂蛋白和 可溶性 TLR2 融合蛋白剖析 TLR2-配体相互作用相关的结构-功能关系 来自 MTB 的这些具有病理生理学重要意义的 TLR2 激动剂。目标 1 将利用细胞因子分泌 用于研究 His 标记的重组 MTB 脂蛋白的活性及其受体依赖性的读数 （使用TLR1或TLR6作为与TLR2异二聚体的共受体，以及使用辅助受体， CD14 和 CD36)。目标 2 将确定影响 MTB 脂蛋白相互作用的结构特征 利用巨噬细胞和树突状细胞检测 TLR2、TLR1、TLR6 和辅助受体（CD14 和 CD36） 来自基因缺陷受体的小鼠并分析 MTB 脂蛋白变体，无需 酰化和/或蛋白质序列中的缺失、截短或突变（或使用最小活性 构建体表达为重组蛋白或制成合成肽）。目标 3 将使用直接 生化结合测定研究标记的重组可溶性 TLR 和脂蛋白分子的结合。 我们将测量不同MTB脂蛋白及其结构变体对TLR2的亲和力，以进一步 了解激动剂与 TLR2 结合的结构决定因素。总体而言，我们将确定结构 MTB 脂蛋白与 TLR2 结合的基础，包括脂质和蛋白质成分的贡献。 相关性：这些研究将为 TLR2 的机制提供独特而新颖的见解。 识别山地车。 TLR2 是参与 MTB 识别的关键免疫系统受体。更大 了解其功能将有助于揭示导致宿主抵抗的重要免疫机制 和/或 MTB 慢性感染期间逃避免疫力。这可能有助于开发更好的治疗方法 结核。它还可能有助于设计更好的免疫佐剂，用于广泛的治疗用途。