TLR-mediated Signaling Complex Formation and Regulation of Effector Functions

TLR 介导的信号复合物的形成和效应器功能的调节

• 批准号: 7696936
• 负责人: HANS HAECKER
• 金额: $ 42万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-23 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7696936
• 关键词: Adaptor Signaling Protein; Affinity Chromatography; Agonist; Antibiotics; Autoimmune Diseases; Bacteria; Bacterial Proteins; Binding; Biochemical; Biological Assay; Biology; Cells; Chimeric Proteins; Communicable Diseases; Complement; Complex; Data; Development; Dimerization; Disease; Epitopes; Event; Family; Family member; Figs - dietary; Future; Gene Expression Regulation; Goals; Human; IRAK1 gene; Immune; Immune Cell Activation; Immune response; In Vitro; Inflammatory; Intervention; Knowledge; Life; Lipopolysaccharides; Lymphoid; Maps; Marshal; Mass Spectrum Analysis; Mediating; Modeling; Molecular; Mus; Nucleotides; Organ Size; Pathology; Pathway interactions; Peptidoglycan; Phenotype; Phosphotransferases; Physiological; Planning Techniques; Procedures; Process; Production; Protein Analysis; Proteins; Proteomics; Receptor Activation; Receptor Signaling; Recruitment Activity; Regulation; Role; Sepsis; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Signaling Molecule; Signaling Protein; Specificity; System; TLR10 gene; TNF receptor-associated factor 3; TRAF6 gene; Techniques; Therapeutic; Therapeutic Intervention; Toll-like receptors; Transduction Gene; Translating; Translations; Virus; activating transcription factor; base; coumermycins; cytokine; fungus; in vivo; macrophage; member; mutant; novel; pathogen; public health relevance; receptor-mediated signaling; research study

DESCRIPTION (provided by applicant): The major goal of this project is to define and characterize unknown components of the signaling pathways triggered by Toll-like receptors (TLRs). The function of TLRs on innate immune cells is to recognize pathogens and translate recognition into cell activation and appropriate effector functions, e.g. cytokine production. Ten TLRs are known in humans that specifically recognize diverse pathogen constituents such as lipopolysaccharides (LPS), peptidoglycans (PGN), nucleotides and proteins. This enormous range of specificities enables innate immune cells to respond immediately to virtually all classes of pathogens, including bacteria, fungi, viruses and protozoans. In addition to their physiological role in immune defense, TLRs are also critically involved in the development of immune pathology as elicited by inappropriate TLR stimulation during sepsis and autoimmune diseases. As such, the TLRs and their signal transduction pathways represent important targets for therapeutic intervention strategies. Several hierarchically acting key components of the TLR signal transduction pathways have been defined: TLR activation leads to recruitment and oligomerization of the adaptor protein MyD88, which binds to members of the IRAK family, which in turn recruit TRAF6. TRAF6 oligomerization induces diverse signaling pathways, eventually leading to activation of downstream effector kinases that directly activate transcription factors. Still, it is unknown whether additional proteins exist that act in between MyD88 and TRAF6. Also, little is known about the signaling events between TRAF6 and many of the downstream effector kinases. One reason for this lack of information is that it is technically difficult to explore transiently assembled signaling complexes. We have developed a robust and sensitive technique that allows purification and characterization of such signaling complexes. In this procedure, TLR-mediated dimerization of signaling proteins is mimicked by fusion of these proteins to the bacterial protein Gyrase B, which can inducibly be dimerized by the bivalent antibiotic coumermycin A1. When fused to MyD88 or TRAF6 and equipped with additional epitope tags, the highly selective signaling complexes can be purified and characterized by mass spectrometry (MS). We have demonstrated the proof of principle for this approach by identifying TRAF3 as a critical component of TLR signaling pathways. We have recently optimized the protein analysis using quantitative MS, allowing precise definition of the dynamic composition of signaling complexes. We now propose to pursue two specific aims: First, to define new components of the key TLR signaling complexes marshaled by MyD88 and TRAF6 using our dimerization technique and, second, to define the function of ABIN-1, a novel TLR signaling component recently discovered by our technique. Preliminary analysis of ABIN-1 deficient mice indicates an inflammatory phenotype with increased lymphoid organ size. PUBLIC HEALTH RELEVANCE: Toll-like receptor family members constitute a major pathogen recognition system, which protects us from infectious diseases via partially characterized signaling pathways. The goal of this project is to reveal the molecular composition of these pathways and to define the function of identified molecules in immune responses. This information will be crucial for our understanding of physiological immune responses, and will also be instrumental for future therapeutic approaches in diseases which are controlled by non-physiological activity of these pathways, i.e. autoimmune diseases and sepsis.

描述（由申请人提供）：该项目的主要目标是定义和表征toll样受体（TLRs）触发的信号通路的未知成分。tlr对先天免疫细胞的作用是识别病原体，并将识别转化为细胞活化和适当的效应功能，如细胞因子的产生。已知人类有10个tlr特异性识别多种病原体成分，如脂多糖（LPS）、肽聚糖（PGN）、核苷酸和蛋白质。这种巨大的特异性范围使先天免疫细胞能够立即对几乎所有类别的病原体作出反应，包括细菌、真菌、病毒和原生动物。除了在免疫防御中的生理作用外，TLR还在败血症和自身免疫性疾病期间不适当的TLR刺激引起的免疫病理发展中起关键作用。因此，tlr及其信号转导通路是治疗干预策略的重要靶点。TLR信号转导途径的几个层次作用的关键组成部分已经被定义：TLR激活导致接头蛋白MyD88的募集和低聚化，MyD88与IRAK家族成员结合，进而募集TRAF6。TRAF6寡聚化诱导多种信号通路，最终导致直接激活转录因子的下游效应激酶的激活。然而，是否存在其他蛋白在MyD88和TRAF6之间起作用尚不清楚。此外，对于TRAF6与许多下游效应激酶之间的信号传导事件知之甚少。这种信息缺乏的一个原因是技术上难以探索瞬时组装的信号复合物。我们已经开发了一种强大而敏感的技术，可以纯化和表征这些信号复合物。在这个过程中，通过将信号蛋白与细菌蛋白Gyrase B融合来模拟tlr介导的信号蛋白二聚化，该蛋白可以被二价抗生素库默霉素A1诱导二聚化。当与MyD88或TRAF6融合并配备额外的表位标签时，高选择性的信号复合物可以通过质谱（MS）纯化和表征。我们通过确定TRAF3是TLR信号通路的关键组成部分，证明了这种方法的原理。我们最近使用定量质谱优化了蛋白质分析，允许精确定义信号复合物的动态组成。我们现在提出追求两个具体目标：首先，使用我们的二聚化技术定义由MyD88和TRAF6编组的关键TLR信号复合物的新组分；其次，定义ABIN-1的功能，ABIN-1是我们技术最近发现的一种新的TLR信号组分。初步分析显示，ABIN-1缺陷小鼠的炎症表型与淋巴器官大小增加有关。公共卫生相关性：toll样受体家族成员构成了一个主要的病原体识别系统，它通过部分特征的信号通路保护我们免受传染病的侵害。该项目的目标是揭示这些途径的分子组成，并确定已识别分子在免疫反应中的功能。这些信息将对我们理解生理免疫反应至关重要，也将有助于未来治疗由这些途径的非生理活动控制的疾病，即自身免疫性疾病和败血症。