M. tuberculosis lipoprotein-TLR2 interactions

描述（由申请人提供）： 我们的目标是确定结核分枝杆菌（MTB）脂蛋白与Toll样受体（TLR）-2结合的生物化学和结构基础以及由此产生的激动剂活性。TLR 2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TLR 2在结核病中具有这种关键作用，但TLR 2识别MTB脂蛋白的结构基础仍然知之甚少。此外，TLR 2在识别其他致病物种中发挥作用，但TLR 2激动剂活性的结构决定因素在很大程度上尚未探索。已知脂蛋白的酰基结构影响其被TLR 2识别，但蛋白质结构对TLR 2结合的影响基本上是未知的。我们已经表征了三种不同的MTB脂蛋白，其通过TLR 2进行信号传导：LpqH（19-kDa脂蛋白）、LprG和LprA。这些脂蛋白都是TLR 2激动剂，但其活性的效力和表观结构决定因素不同。我们的数据表明，MTB脂蛋白的脂质和蛋白质组分都可以影响TLR 2激动剂活性。我们正在构建重组标记的脂蛋白和可溶性TLR 2融合蛋白，以剖析TLR 2-配体相互作用中的结构-功能关系，这些相互作用与MTB的这些病理生理学上重要的TLR 2激动剂有关。目的1将使用细胞细胞因子分泌读数来研究His标记的重组MTB脂蛋白的活性及其受体依赖性（使用TLR 1或TLR 6作为具有TLR 2的异源二聚体中的共受体，以及使用辅助受体CD 14和CD 36）。目的二是确定结核分枝杆菌脂蛋白的结构特征，从而影响与TLR 2、TLR 1、TLR 6和辅助受体的相互作用（CD 14和CD 36），并分析没有酰化和/或缺失的MTB脂蛋白变体，蛋白质序列中的截短或突变（或使用表达为重组蛋白或制备为合成肽的最小活性构建体）。目的3将使用直接生物化学结合试验来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TLR 2的亲和力，以进一步了解激动剂与TLR 2结合的结构决定因素。总之，我们将确定MTB脂蛋白结合TLR 2的结构基础，包括脂质和蛋白质组分的贡献。 相关性：这些研究将为TLR 2识别MTB的机制提供独特和新颖的见解。TLR 2是参与MTB识别的关键免疫系统受体。更深入地了解其功能将有助于揭示在MTB慢性感染期间导致宿主抵抗和/或免疫逃避的免疫中的重要机制。这可能有助于开发更好的结核病治疗方法。它还可以帮助设计更好的免疫佐剂，用于广泛的治疗用途。

DESCRIPTION (provided by applicant): 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 variants without 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.