M. tuberculosis lipoprotein-TLR2 interactions

描述(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结合的生化和结构基础以及由此产生的激动剂活性。TLR2识别结核分枝杆菌脂蛋白启动天然免疫并影响对结核分枝杆菌的获得性免疫。尽管TLR2在结核病中发挥了关键作用，但TLR2识别结核分枝杆菌脂蛋白的结构基础仍然知之甚少。此外，TLR2还具有识别其他病原物种的功能，但TLR2激动剂活性的结构决定因素在很大程度上尚不清楚。已知脂蛋白的酰基结构影响TLR2对其的识别，但蛋白质结构对TLR2结合的影响基本上是未知的。我们已经鉴定了三种不同的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，但在效力和其活性的明显结构决定因素上有所不同。我们的数据表明，MTB脂蛋白的脂质和蛋白质成分都可以影响TLR2激动剂的活性。我们正在构建重组标记脂蛋白和可溶性TLR2融合蛋白，以剖析与MTB中这些重要的TLR2激动剂相关的TLR2-配体相互作用中的结构-功能关系。目的1利用细胞细胞因子分泌读数研究His标记的重组MTB脂蛋白的活性及其受体依赖性(使用TLR1或TLR6作为与TLR2的异源二聚体的共同受体，以及使用辅助受体CD14和CD36)。目的通过利用TLR2、TLR1、TLR6及其受体(CD14和CD36)受体的遗传缺陷小鼠的巨噬细胞和树突状细胞，以及分析无酰化和/或蛋白质序列缺失、截断或突变的MTB脂蛋白变体(或使用表达为重组蛋白或合成多肽的最低活性构建体)，来确定影响与TLR2、TLR1、TLR6和辅助受体(CD14和CD36)相互作用的MTB脂蛋白的结构特征。目的3利用直接生化结合实验研究标记的重组可溶性TLR与脂蛋白分子的结合。我们将测量不同MTB脂蛋白及其结构变体与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MTB脂蛋白与TLR2结合的结构基础，包括脂质和蛋白质组分的贡献。 相关性：这些研究将为TLR2识别结核分枝杆菌的机制提供独特和新颖的见解。TLR2是参与识别结核分枝杆菌的关键免疫系统受体。对其功能的进一步了解将有助于揭示在MTB慢性感染过程中导致宿主抵抗和/或免疫逃避的重要免疫机制。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.