TLR Signaling and Protein Fragment Complementation

TLR 信号传导和蛋白质片段互补

• 批准号: 6758801
• 负责人: PETER S TOBIAS
• 金额: $ 23.46万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6758801
• 关键词: beta lactamase; biological signal transduction; enzyme activity; fluorescence; immunoprecipitation; molecular assembly /self assembly; protein protein interaction; proteins; receptor binding; toll like receptor

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.

描述（由申请人提供）：受体是告知细胞内部外部发生什么的分子。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物的组装或激活。在这个建议中，我们概述了使用一种新的方法来定义TLR受体组装。TLR现在被广泛认为是各种各样的配体（外源性以及内源性）的检测器。它们的激活启动先天免疫炎症反应，并促进适应性免疫反应。积极的一面是，它们对检测感染至关重要，消极的一面是，它们与败血性休克有关。 了解它们的激活机制和细胞内信号传导对于理解，甚至改变它们激活的后果是很重要的。激活它们的替代方法也可能有价值，也许是作为新的佐剂。典型的，有用的，了解大会的信号复合物的细胞内结构域的受体已经变化，他们包括这样的方法，如酵母双杂交系统，共免疫沉淀，定点诱变。对于这一组，使用TLR的背景下，我们建议添加蛋白质片段互补。许多蛋白质的选定片段可以缔合以产生功能性双分子复合物。在一种方法中，当酶的无活性片段以一定方向聚集在一起时，产生酶活性，使得它们可以重组并催化可检测产物的形成。鉴于酶反应的催化性质，这种方法可以非常灵敏。在另一种方法中，荧光蛋白的片段在适当地聚集在一起时变得发荧光。我们建议开发的酶是β-内酰胺酶，我们建议开发的荧光蛋白是黄色荧光蛋白（YFP）。因此，我们有两个目标：使用免疫共沉淀、β-内酰胺酶和YFP片段互补来定义TLR系统中的新型蛋白质-蛋白质相互作用，并对互补系统进行改变以提高其用于我们目的的效用。