M. tuberculosis lipoprotein-TLR2 interactions

结核分枝杆菌脂蛋白-TLR2 相互作用

• 批准号: 7994850
• 负责人: Clifford V Harding
• 金额: $ 37.86万
• 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 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7994850
• 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; 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; adaptive immunity; base; cytokine; design; insight; loss of function; macrophage; novel; pathogen; programs; protein structure; receptor; receptor function; research study; response; synthetic peptide; tool; tuberculosis immunity; 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合结核病的生化和结构基础 （MTB）脂蛋白到Toll样受体（TLR）-2和产生的激动剂活性。 TLR2识别MTB 脂蛋白启动先天免疫，并影响对MTB的适应性免疫。尽管这一关键作用 TLR2在结核病中，TLR2识别MTB脂蛋白的结构基础仍然很差 理解。另外，TLR2在识别其他致病物种方面起作用，但结构 TLR2激动剂活性的决定因素在很大程度上没有探索。众所周知，脂蛋白的酰基结构 影响了TLR2的识别，但是蛋白质结构对TLR2结合的影响本质上是 未知。我们表征了三种不同的MTB脂蛋白，它们通过TLR2发出信号：LPQH（19-kDa 脂蛋白），LPRG和LPRA。这些脂蛋白都是TLR2激动剂，但在效力和明显方面有所不同 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均 脂蛋白会影响TLR2激动剂活性。我们正在构建重组标记的脂蛋白和 可溶性TLR2融合蛋白剖析TLR2-rigand相互作用中的结构功能关系 对于这些在病理生理上重要的TLR2激动剂中，来自MTB。 AIM 1将使用细胞因子分泌 读数以研究His标记的重组MTB脂蛋白及其受体依赖性的活性 （将TLR1或TLR6用作具有TLR2的异二聚体中的共受体，以及使用辅助受体的使用， CD14和CD36）。 AIM 2将确定MTB脂蛋白的结构特征，影响与 TLR2，TLR1，TLR6和辅助受体（CD14和CD36）通过使用巨噬细胞和树突状细胞 来自遗传缺乏的小鼠，其中MTB脂蛋白变体的受体和分析 蛋白质序列中的缺失，截断或突变（或使用最小活性 以重组蛋白或合成肽形式表示的构建体。 AIM 3将使用直接 生化结合测定法研究了标记的重组可溶性TLR和脂蛋白分子的结合。 我们将测量不同MTB脂蛋白的亲和力及其对TLR2的结构变体的进一步 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MTB。 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免疫力的重要机制，从而导致宿主抵抗 MTB慢性感染期间对免疫的逃避。这可能有助于开发更好的治疗方法 结核。它也可能有助于设计更好的免疫佐剂，以用于多种治疗用途。