Investigation of Systems Regulation in TLR Signaling

描述（由申请人提供）：尽管通过toll样受体（TLR）的免疫应答保护细胞免受毒性损伤，但如果不加以控制，病原体刺激的细胞因子过度产生可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识别特定病原体产物的信号机制，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。基于我们的发现，在LPS刺激的巨噬细胞中，一种新的TLR 4相互作用蛋白作为NF κ B活化的差异调节剂发挥作用，我们假设许多功能未知的TLR下游蛋白以合作和及时的方式差异调节TLR信号的开启和关闭。通过使用我们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实时系统地识别TLR介导的通路。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）沿着激动剂特异性TLR信号转导中继进行多蛋白信号复合物的“路径尺度”分析。我们的“双标记”分析整合了天然复合物形成、表位亲和力分离和系统规模研究所需的“光谱内”定量测量的能力，所述系统规模研究将用于从各种激动剂刺激的活巨噬细胞分离的“双标记”含诱饵复合物的综合分析，（2）对激动剂特异性TLR介导的途径中的信号调节中的新信号蛋白的功能作用/连接进行“途径尺度”表征。新的蛋白质/它们的相互作用的功能和机制表征将揭示在激动剂特异性刺激后通过它们的下游相互作用体选择性调节TLR介导的信号的新的光，和（3）表征与疾病发病机制相关的TLR协同作用相关的多TLR介导的信号相互作用体。病原体可能含有几种TLR激动剂，这些激动剂触发多种TLR介导的信号传导途径，通过不适当地产生炎性细胞因子协同促进疾病发病机制。我们的系统方法具有独特的优势，可以解决信号相互作用组在操作多途径协调和“交叉对话”中的复杂性。总之，对负责选择性信号调节的信号相互作用组的分子理解将为人类炎症性疾病的控制提供新的见解。

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.