Initiation of Toll-like Receptor Signaling

描述（由申请人提供）：先天免疫系统执行立即保护宿主免受感染并产生警报和指导获得性免疫作用的反应的关键任务。Toll样受体（TLR）是先天免疫的基本要素，其在直接识别微生物、真菌和病毒组分后激活细胞事件，导致炎症、直接杀死病原体和增强的抗原呈递。它们在诱导和调节这些事件中的重要作用通过它们与多种免疫病症的直接关联来证明，所述免疫病症包括败血症、动脉粥样硬化、关节炎、哮喘和自身免疫。在TLR家族的10个成员中，TLR 2需要TLR 1或TLR 6来识别、区分和启动对多种抗菌成分的应答。宿主反应由两种细胞质近端衔接分子（MyD 88和TIRAP/MAL）介导。该项目的长期目标是确定该TLR亚家族识别其激动剂、启动细胞反应并协调这些反应的机制。在亚细胞和分子水平上的反应。具体目的是1）确定TLR 1和6在识别微生物和真菌组分以及所涉及的受体的细胞外区域中的精确作用，2）确定在激动剂介导的细胞活化后TLR 1、2和6与近端衔接分子MyD 88和TIRAP/ MAL之间的细胞内运输和物理相互作用，和3）确定近端信号传导事件对观察到的TLR的细胞表面共定位和其它TLR相关的内吞运输事件的作用。TLR的特定功能，激动剂歧视以及这种歧视的结构基础，通过使用合成的激动剂和受体结构域交换实验进行评估。TLR和衔接子的定位通过直接显微镜进行评估，包括FRET、膜分离和药理学试剂。显性负性分子、shRNA和药理学试剂的作用解决了信号传导事件对细胞内TLR运输的作用。

DESCRIPTION (provided by applicant): The innate immune system performs the critical task of immediately protecting the host from infection and generating responses that alert and guide the actions of acquired immunity. Toll-like receptors (TLRs) are essential elements of innate immunity which upon direct recognition of microbial, fungal and viral components activate cellular events leading to inflammation, direct killing of the pathogen, and enhanced antigen presentation. Their essential role in inducing and regulating these events, is evidenced by their direct association with a variety of immune disorders ranging from sepsis, atherosclerosis, arthritis, asthma and autoimmunity. Among the ten member TLR family, TLR2 requires TLR1 or TLR6 to recognize, discriminate and initiate responses to a wide variety of rnicrobial components. Host responses are mediated by the two cytoplasmic proximal adaptor molecules known as MyD88 and TIRAP/MAL. The long term goal of this project is to define the mechanism by which this subfamily of TLRs recognize their agonists, initiate cellular responses, and coordinate these responses at the subcellular and molecular level. The specific aims are 1) To define the precise role of TLRs 1 and 6 in recognition of microbial and fungal components as well as the extracellular regions of the receptors involved, 2) To determine the intracellular trafficking and physical interactions among TLRs 1, 2 and 6 and the proximal adaptor molecules MyD88 and TIRAP/ MAL upon agonist-mediated cell activation, and 3) To define the role of proximal signaling events; on the observed cell surface colocalization of TLRs and other TLR-associated endocytic trafficking events. The specific function of TLRs, with respect to agonist discrimination as well as the structural basis of this discrimination, are assessed through the use of synthetic; agonists and receptor domain swapping experiments. The localization of TLRs and adaptors are assessed through direct microscopy including FRET, membrane fractionation, and pharmacalogic agents. The effect of dominant negative molecules, shRNA and pharmacalogic agents address the role of signaling events on intracellular TLR trafficking.