TLR Signaling and Protein Fragment Complementation

描述(申请人提供)：受体是告知细胞内部正在发生什么的分子。细胞表面受体的功能有三个关键步骤。第一种是配体结合，第二种是跨膜信号转导，第三种是细胞内信号起始复合体的组装或激活。在这项提案中，我们概述了使用一种新的方法来定义TLR受体组装。TLRs现在被广泛认为是各种配体的检测器，无论是外源性的还是内源性的。它们的激活启动了先天免疫炎症反应，也促进了获得性免疫反应。从积极的方面来说，它们对检测感染至关重要，而在消极的方面，它们与感染性休克有关。 了解它们的激活机制和细胞内信号对于理解甚至改变它们的激活后果很重要。激活它们的替代方法也可能有价值，也许是作为新的佐剂。了解受体胞内结构域组装信号复合体的典型而有用的方法有多种，包括酵母双杂交系统、免疫共沉淀和定点突变等方法。对于这一组，利用TLRs的背景，我们建议增加蛋白质片段互补。许多蛋白质的选定片段可以结合在一起，形成功能性的双分子复合体。在一种方法中，当酶的非活性片段以一定的方向聚集在一起，使得它们可以重组并催化形成可检测的产物时，就产生了酶活性。考虑到酶反应的催化性质，这种方法可能非常敏感。在另一种方法中，当适当地结合在一起时，荧光蛋白的片段就会变成荧光。我们要开发的酶是β-内酰胺酶，我们要开发的荧光蛋白是黄色荧光蛋白(YFP)。因此，我们有两个目标：利用免疫共沉淀、β-内酰胺酶和YFP片段互补来定义TLR系统中新的蛋白质-蛋白质相互作用，并对互补系统进行改造，以提高它们的实用性。

DESCRIPTION (provided by applicant): Receptors are the molecules that inform the inside of a cell what is going on outside. There are three critical steps to the function of a cell surface receptor. The first is ligand binding, the second is transmembrane signal transduction, the third is assembly or activation of an intracellular signal initiation complex. In this proposal we outline the use of a novel approach to define TLR receptor assembly. TLRs are now widely appreciated as detectors of a broad variety of ligands, exogenous as well as endogenous. Their activation initiates innate immune inflammatory responses and promotes adaptive immune responses as well. On the positive side, they are critical for detection of infection and on the negative, are involved in septic shock. Understanding their mechanisms of activation and intracellular signaling is important for understanding, and perhaps altering, the consequences of their activation. Alternate means of activating them could also have value, perhaps as novel adjuvants. Typical, and useful, approaches to understanding assembly of a signaling complex by the intracellular domain of a receptor have been varied; they include such approaches as the yeast two-hybrid system, coimmunoprecipitation, and site directed mutagenesis. To this group, using the context of TLRs, we propose to add protein fragment complementation. Selected fragments of many proteins can associate to produce functional bimolecular complexes. In one approach an enzyme activity is generated when inactive fragments of an enzyme are brought together in an orientation such that they can recombine and catalyze the formation of a detectable product. Given the catalytic nature of enzyme reactions, this approach can be remarkably sensitive. In another approach fragments of fluorescent proteins become fluorescent when appropriately brought together. The enzyme we propose to exploit is beta-Iactamase and the fluorescent protein we propose to exploit is yellow fluorescent protein (YFP). Thus we have two goals: To define novel protein-protein interactions in the TLR system using co-immunoprecipitation, beta- lactamase and YFP fragment complementation and to make alterations to the complementation systems to improve their utility for our purposes.