TLR-mediated Signaling Complex Formation and Regulation of Effector Functions

描述(由申请人提供)：该项目的主要目标是定义和表征由Toll样受体(TLRs)触发的信号通路的未知成分。TLRs在天然免疫细胞上的功能是识别病原体，并将识别转化为细胞激活和适当的效应功能，如细胞因子的产生。已知有10种TLRs在人类中专门识别不同的病原体成分，如脂多糖(LPS)、肽多聚糖(PGN)、核苷酸和蛋白质。这种巨大的特异性使先天免疫细胞能够立即对几乎所有类别的病原体做出反应，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，TLRs还在脓毒症和自身免疫性疾病中由不适当的TLR刺激引起的免疫病理的发生发展中起关键作用。因此，TLRs及其信号转导通路是治疗干预策略的重要靶点。已经确定了TLR信号转导通路的几个分级作用的关键成分：TLR激活导致接头蛋白MyD88的招募和寡聚化，MyD88与IRAK家族成员结合，进而招募TRAF6。TRAF6寡聚诱导不同的信号通路，最终导致下游效应激酶的激活，从而直接激活转录因子。然而，目前尚不清楚是否存在其他蛋白质在MyD88和TRAF6之间发挥作用。此外，对TRAF6和许多下游效应器激酶之间的信号事件也知之甚少。这种缺乏信息的原因之一是，在技术上很难探索瞬时组装的信号复合体。我们已经开发了一种强大而灵敏的技术，可以对这种信号复合体进行纯化和表征。在这个过程中，TLR介导的信号蛋白的二聚化是通过将这些蛋白融合到细菌蛋白旋转酶B上来模拟的，细菌蛋白旋转酶B可以被双价抗生素Coumermycin 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体可以被纯化并用质谱仪(MS)进行表征。我们已经通过将TRAF3确定为TLR信号通路的关键组成部分来证明这一方法的原则证据。我们最近使用定量MS优化了蛋白质分析，允许精确定义信号复合体的动态组成。我们现在建议追求两个特定的目标：第一，使用我们的二聚化技术定义由MyD88和TRAF6编组的关键TLR信号复合体的新组件；第二，定义ABIN-1的功能，ABIN-1是我们的技术最近发现的一个新的TLR信号组件。对ABIN-1缺陷小鼠的初步分析表明，淋巴器官大小增加是一种炎性表型。公共卫生相关性：Toll样受体家族成员构成一个主要的病原体识别系统，该系统通过部分特征化的信号通路保护我们免受传染病的侵袭。该项目的目标是揭示这些途径的分子组成，并确定已识别的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。

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.