DEFINING INNATE IMMUNE RECEPTOR LIGAND BINDING SITES

描述（由申请人提供）：先天免疫系统显示一系列模式识别受体（PRRs），用于检测微生物上的病原体相关分子模式（PAMPs）。最近的工作已经确定toll样受体是PRR家族的重要成员。这些受体在检测到病原微生物时启动防御反应。在大多数情况下，这些反应导致感染的解决，但在其他情况下，它们导致有害的炎症反应，如急性肺损伤或可能的慢性疾病炎症。本研究的目的是了解PAMPs受体识别的分子基础。具体来说，我们提出了两个假设：1)PAMP与其同源PRRs直接相互作用；2)PRRs具有多个PAMP识别位点。为了研究这些假设，我们制定了四个具体目标：SAI：合成相关先天免疫系统PRRs的特异性交联PAMPs。SA 2：利用可交联配体测试PAMPs和PRRs之间的直接相互作用，并通过质谱定义交联位点来确定PRRs与PAMPs的特定附着位点。SA 3：通过确定给定PAMP与其提议的PRR的结合（或交联）是否与该PRR特异性的其他PAMP组竞争或独立，来测试特定PRR上存在多个PAMP结合位点。SA 4：设计突变策略来测试a) Specific Aim 2中确定的交联位点在配体结合和细胞活化中是否具有重要的功能，b)单个PRRs上是否存在不同PAMPs的多个结合位点。通过了解PRRs如何识别PAMP，我们希望能够提出治疗性操纵PAMP - PRR相互作用的方法。

DESCRIPTION (provided by applicant): The innate immune system displays a series of Pattern Recognition Receptors (PRRs) that detect Pathogen Associated Molecular Patterns (PAMPs) on microorganisms. Recent work has identified the Toll-like receptors as important members of the PRR family. These receptors initiate defensive responses upon detection of pathogenic microorganisms. In most cases, these responses lead to resolution of infection but in others, they lead to deleterious inflammatory responses such as acute lung injury or possibly the inflammation of chronic diseases. The goal of this research is to understand the molecular basis for receptor recognition of PAMPs. Specifically, we propose two hypotheses: 1) that PAMPs interact directly with their cognate PRRs, and 2) that PRRs have more than one PAMP recognition site. To study these hypotheses we have formulated four specific aims: SAI: To synthesize specific cross linkable PAMPs for the relevant innate immune system PRRs. SA 2: To utilize cross linkable ligands to test for direct interactions between the PAMPs and the PRRs and to determine the specific sites of attachment of the PRRs to the PAMPs by mass spectrometric definition of the cross linking sites. SA 3: To test for the existence of multiple PAMP binding sites on a specific PRR by determining whether binding (or cross linking) of a given PAMP to its proposed PRR is competitive with or independent of a panel of other PAMPs specific for that PRR. SA 4: To devise mutational strategies to test a) whether sites of cross linking determined in Specific Aim 2 are functionally important in ligand binding and cellular activation and b) whether there are multiple binding sites for different PAMPs on single PRRs. By understanding how PRRs recognize PAMPs we hope to be able to propose methods for therapeutic manipulation of PAMP - PRR interactions.