M. tuberculosis lipoprotein-TLR2 interactions

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的结合以及产生的激动剂活性。MTB的TLR 2识别 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， TLR 2在结核病中，TLR 2识别MTB脂蛋白的结构基础仍然很差 明白此外，TLR 2在识别其他致病性物种中起作用，但其结构上与其他致病性物种不同。 TLR 2激动剂活性的决定因素在很大程度上未被探索。已知脂蛋白的酰基结构 影响它们被TLR 2识别，但蛋白质结构对TLR 2结合的影响基本上是 未知我们已经鉴定了三种不同的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，但在效力和表观性质上不同。 其活动的结构决定因素。我们的数据表明MTB的脂质和蛋白质组分 脂蛋白可以影响TLR 2激动剂活性。我们正在构建重组标记脂蛋白， 可溶性TLR 2融合蛋白以剖析TLR 2-配体相互作用相关的结构-功能关系 这些病理生理学上重要的TLR 2激动剂。目的1将利用细胞因子分泌 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 (use TLR 1或TLR 6作为与TLR 2的异二聚体中的共受体，以及辅助受体的用途， CD 14和CD 36）。目的2将确定影响与MTB相互作用的MTB脂蛋白的结构特征。 TLR 2、TLR 1、TLR 6和辅助受体（CD 14和CD 36），使用巨噬细胞和树突状细胞 从这些受体遗传缺陷的小鼠和MTB脂蛋白变体的分析， 酰化和/或在蛋白质序列中具有缺失、截短或突变（或使用最小的活性 表达为重组蛋白或制备为合成肽的构建体）。目标3将直接使用 生物化学结合测定以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，以进一步确定MTB脂蛋白及其结构变体对TLR 2的亲和力。 了解与TLR 2结合的激动剂的结构决定因素。总体上，我们将确定 MTB脂蛋白与TLR 2结合的基础，包括脂质和蛋白组分的贡献。 相关性：这些研究将提供独特的和新颖的见解的机制，TLR 2 识别MTB。TLR 2是参与MTB识别的关键免疫系统受体。更大 了解它的功能将有助于揭示免疫中导致宿主抗性的重要机制 和/或在MTB慢性感染期间免疫逃避。这可能有助于开发更好的治疗方法， 结核它还可以帮助设计更好的免疫佐剂，用于广泛的治疗用途。

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.