A Quantitative Proteomic Study of MyD88 Pathways

MyD88 通路的定量蛋白质组学研究

• 批准号: 7197640
• 负责人: TONY WANG
• 金额: $ 18.56万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7197640
• 关键词: Quantitative; Proteomic; Study; MyD88; Pathways

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（由申请人提供）：MyD88是除TLRs外所有哺乳动物Toll样受体（TLRs）介导途径的关键衔接蛋白。我们的长期目标是表征由tlr介导的先天免疫反应的信号网络，作为开发潜在药物靶点的必要前提，以改变通过tlr激活先天免疫的后果，这一领域已被列为NIAID优先研究领域。该提案的中心假设是MyD88可能与不同的蛋白质相互作用，从而在TLR2和TLR9激活后触发不同的途径。我们基于以下观察得出这一假设：(1)TLRs的一个子集，TLR7、TLRs和TLR9，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作为适配器，但不会导致IFN-?的产生。(2) TLR9信号导致IFN-？依赖于MyD88-IRF-7的相互作用，这种相互作用仅发生在核内体囊泡中。(3) MyD88能够结合仅参与特定TLR通路的蛋白。了解免疫信号复合物组装的典型方法包括酵母双杂交系统、通过计算分析的蛋白质同源物搜索、共免疫沉淀和定点诱变等方法。然而，上述方法在直接从实际免疫细胞中识别信号复合物成分方面效率相对较低。我们之前开发了一种基于定量质谱的方法，可以在TLR4激动剂刺激后直接从小鼠巨噬细胞中超灵敏地检测MyD88相互作用蛋白。在本提案中，我们计划进一步改进该技术，以便检测和表征TLR2和TLR9激活后的MyD88信号复合物。因此，我们有两个目标：一是进一步完善免疫信号研究的定量蛋白质组学方法；二是通过直接鉴定MyD88周围在触发不同TLR通路时形成的相互作用蛋白，明确MyD88介导的不同TLR通路的信号特异性。通过这些学习，将获得的知识。为对信号研究感兴趣的生物学家开发一种高通量技术。