Investigation of Systems Regulation in TLR Signaling

描述（由申请人提供）：虽然通过toll样受体（TLRs）的免疫反应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tlr4相互作用蛋白作为NFkappaB激活的差异调节剂，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出tlr介导的信号通路。为了解决疾病发病相关信号蛋白相互作用网络（相互作用组）的复杂性，我们计划：(1)沿着激动剂特异性TLR信号转导继电器对多蛋白信号复合物进行“途径尺度”分析。我们的“双标记”分析整合了自然复合物形成、表位亲和分离和系统规模调查所需的“光谱内”定量测量的能力，将对从各种激动剂刺激的活巨噬细胞中分离的“双标记”含诱饵复合物进行全面分析。(2)在激动剂特异性TLR介导的信号调节途径中，对新型信号蛋白的功能角色/联系进行“通路尺度”表征。新蛋白/其相互作用的功能和机制表征将为TLR介导的信号在激动剂特异性刺激后通过其下游相互作用组募集的选择性调节提供新的线索；(3)表征与疾病发病相关的TLR协同相关的多TLR介导的信号相互作用组。病原体可能含有多种TLR激动剂，可触发多种TLR介导的信号通路，通过不适当的炎症细胞因子产生协同促进疾病发病。我们的系统方法在解决多通路协调和“交叉对话”操作中信号相互作用组的复杂性方面具有独特的优势。综上所述，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.