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A Quantitative Proteomic Study of MyD88 Pathways

MyD88 通路的定量蛋白质组学研究

基本信息

批准号：
7365114
负责人：
TONY WANG
金额：
$ 21.85万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-03-01 至 2010-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7365114
关键词：
Adaptor Signaling Protein Agonist Antiviral Response Area Binding Proteins Biological Assay Cell Line Cells Complex Computer Analysis Coupled Dendritic Cells Detection Development Drug Delivery Systems Electrospray Ionization Endosomes Genetic Transcription Goals Hand Homologous Gene Immune Immune response Interferon-alpha Interferon-beta Interferons Knock-out Knowledge Lead Ligands Light Link Lipids Liquid Chromatography Luciferases Macrophage Activation Mass Spectrum Analysis Mediating Methods Molecular Mus Names National Institute of Allergy and Infectious Disease Nature Outcome Pathway interactions Pattern Play Process Production Proteins Proteome Proteomics Receptor Signaling Recruitment Activity Reporter Research Research Personnel Role Set protein Signal Pathway Signal Transduction Site-Directed Mutagenesis Specificity Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization TLR2 gene TLR3 gene TLR4 gene TLR7 gene Technology Testing Time Toll-Like Receptor Pathway Toll-like receptors Validation Vesicle attenuation base cell type high throughput analysis high throughput technology improved interest macrophage mass spectrometer novel pathogen programs receptor response transcription factor two-dimensional yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): MyD88 is the critical adaptor protein for the entire set of mammalian Toll like receptors (TLRs) mediated pathways except TLRS. Our long-term goal is to characterize the signaling network of innate immune responses mediated by TLRs as a necessary prerequisite to the development of potential drug targets to alter the consequences of innate immune activation through TLRs, an area that has been listed as an NIAID prioritized research area. The central hypothesis of this proposal is that MyD88 may interact with different proteins in order to trigger different pathways following TLR2 and TLR9 activation. We base this hypothesis on the following observations: (1) A subset of TLRs, TLR7, TLRS and TLR9, induces antiviral responses by producing interferon-alpha. Activation of TLR2, on the other hand, utilizes MyD88 as adaptor yet does not lead to the production of IFN-?. (2) TLR9 signaling leading to the production of IFN-? is dependent on MyD88-IRF-7 interaction that only occurs in endosome vesicles. (3) MyD88 is capable of binding proteins that are only involved in a specific TLR pathway. Typical approaches to understand assembly of an immune signaling complex include such approaches as the yeast two-hybrid system, protein homologue search by computational analysis, coimmunoprecipitation, and site directed mutagenesis. However, the above approaches are relatively inefficient in direct identification of components of signaling complexes from actual immune cells. We have previously developed a quantitative mass spectrometry-based approach that allowed ultra sensitive detection of MyD88 interacting proteins directly from murine macrophages following stimulation with TLR4 agonist. In this proposal, we plan to further improve this technology so that MyD88 signaling complex following activation of TLR2 and TLR9 can be detected and characterized. Thus we have two goals: First, to further improve the quantitative proteomic approach for immune signaling study; second, to define the signaling specificity of different TLR pathways mediated by MyD88 by directly identifying interacting proteins formed around MyD88 when different TLR pathways are triggered. Through these studies, knowledge of will be gained. A high throughput technology will also be developed for biologists who are interested in signaling study.

描述(申请人提供)：MyD88是哺乳动物Toll样受体(TLRs)介导的整个通路的关键适配子蛋白，但TLRs除外。我们的长期目标是表征TLRs介导的先天性免疫反应的信号网络，作为开发潜在药物靶点以改变通过TLRs激活天然免疫的后果的必要先决条件，这一领域已被列为NIAID优先研究领域。这一提议的中心假设是，MyD88可能与不同的蛋白质相互作用，以便在TLR2和TLR9激活后触发不同的途径。我们基于以下观察结果提出这一假设：(1)TLR7、TLR7和TLR9的一个子集，通过产生干扰素-α诱导抗病毒反应。另一方面，TLR2的激活利用MyD88作为接头，但不会导致干扰素-β的产生。(2)TLR9信号导致干扰素-？依赖于MyD88-IRF-7的相互作用，而这种相互作用只发生在内吞体囊泡中。(3)MyD88能够结合仅参与特定TLR途径的蛋白。了解免疫信号复合体组装的典型方法包括酵母双杂交系统、通过计算分析进行蛋白质同源搜索、免疫共沉淀和定点突变等方法。然而，上述方法在直接从实际免疫细胞中识别信号复合体的成分方面效率相对较低。我们之前已经开发了一种基于定量质谱学的方法，允许在TLR4激动剂刺激后直接从小鼠巨噬细胞中超灵敏地检测MyD88相互作用的蛋白。在这项提案中，我们计划进一步改进这项技术，以便能够检测和表征TLR2和TLR9激活后的MyD88信号复合体。因此，我们有两个目标：第一，进一步完善免疫信号研究的定量蛋白质组学方法；第二，通过直接鉴定当不同的TLR通路被触发时，MyD88周围形成的相互作用的蛋白质，来确定MyD88介导的不同TLR通路的信号特异性。通过这些研究，将获得关于的知识。还将为对信号转导研究感兴趣的生物学家开发高通量技术。