Investigation of Systems Regulation in TLR Signaling

描述(申请人提供)：尽管通过Toll样受体(TLRs)的免疫反应保护细胞免受致命损害，但如果不加以控制，病原体刺激的细胞因子过度产生可能导致器官衰竭和各种严重的炎症性疾病。我们的长期目标是阐明TLR的信号机制，识别特定的病原体产物，同时通过激动剂特异性和协同TLR信号通路控制细胞因子的产生(S)。根据我们的发现，在内毒素刺激的巨噬细胞中，一种新的TLR4相互作用蛋白作为NFkappaB激活的差异调节因子，我们假设许多未知功能的TLR下游蛋白以协作和及时的方式差异地调节TLR信号的开关。通过使用我们最新集成的系统技术平台，我们已经证明，这些新的信号蛋白可以在实际免疫细胞中以TLR介导的途径为特征进行实时系统识别。为了解决疾病发病机制相关的信号蛋白相互作用网络(Interactomes)的复杂性，我们计划：(1)沿着激动剂特异的TLR信号转导继电器对多蛋白信号复合体进行“路径规模”的剖析。我们的“双标记”分析集成了自然复合体形成、表位亲和力分离和系统规模研究所需的“谱内”定量测量能力，该研究将用于全面分析从各种激动剂刺激的活巨噬细胞中分离出的含有“双标记”诱饵的复合体；(2)在激动剂特异性TLR介导的途径中，对新信号蛋白在信号调制中的功能角色/链接进行“途径尺度”表征。新的蛋白质及其相互作用的功能和机制特征将为进一步研究激动剂特异性刺激后招募的下游相互作用体选择性调节TLR介导的信号提供新的线索，以及(3)表征与疾病发病机制相关的TLR协同作用的多TLR介导的信号相互作用组。病原体可能包含几种TLR激动剂，它们触发多个TLR介导的信号通路，通过不适当地产生炎性细胞因子而协同作用于疾病的发病机制。我们的系统方法具有独特的优势，可以解决复杂的信号相互作用组在操作多路径协调和‘串话’。综上所述，对负责选择性信号调制的信号相互作用的分子理解将为控制人类炎症性疾病提供新的视角。

DESCRIPTION (provided by applicant): Although the immune responses via toll-like receptors (TLRs) protect cells from virulent damages, if left unchecked, pathogen-stimulated excessive production of cytokines can lead to organ failures and various severe inflammatory diseases. Our long-term goal is to elucidate the signal mechanisms underlying TLR recognizing specific pathogen products and meanwhile controlling cytokine production through both agonist- specific and synergistic TLR signaling pathway(s). Base on our findings that in the LPS-stimulated macrophages a novel TLR4-interacting protein functions as a differential regulator of NFkappaB activation, we hypothesize that many TLR downstream proteins of unknown function differentially regulate the switching on & off of TLR signaling in a cooperative and timely manner. By using our newly integrated systemic technology platform, we have shown that these novel signal proteins can be systematically identified characterized in a TLR-mediated pathway in actual immune cells in real-time. To resolve the complexity of the disease pathogenesis-associated signal protein interaction networks (interactomes), we plan to: (1) Conduct the 'pathway-scale' profiling of the multiprotein signal complexes along agonist-specific TLR signal transduction relays. Our 'dual-tagging' profiling integrates the capabilities of natural complex formation, epitope affinity isolation, and 'in-spectra' quantitative measurements required for system-scale investigation that will be performed for comprehensive analyses of 'dual-tagged' bait-containing complexes isolated from various agonist-stimulated living macrophages, (2) Perform a 'pathway-scale' characterization of the functional roles/links of novel signal proteins in signal modulation in agonist-specific TLR- mediated pathways. The function and mechanistic characterization of novel proteins/their interactions will shed new light on selective regulation of TLR-mediated signals by their downstream interactome recruited following agonist-specific stimulations, and (3) Characterize the multi-TLR-mediated signaling interactome correlated to disease pathogenesis-related TLR synergy. Pathogens may contain several TLR agonists that trigger multiple TLR-mediated signaling pathways, synergistically contributing to disease pathogenesis through improper production of inflammatory cytokines. Our systemic approach has a unique strength to resolve the complexity of the signaling interactome in operating multi-pathway coordination and 'cross- talking'. Taken together, the molecular understanding of the signal interactomes responsible for selective signal modulation will provide a new insight into the control of human inflammatory diseases.