Endosomal TLRs and their accessory proteins: cell biology and biochemistry

描述(由申请人提供)：以TLR9为例，对内体TLR运输行为的详细了解应有助于设计策略，以改进向专业抗原提呈细胞中的处理隔室递送抗原，并伴随着抗原提呈细胞的适当激活。因此，这些知识有望有助于理解目前正在使用的疫苗的效力，以及如何改进它们。在这方面，B细胞是一个特殊的靶标，因为它们不仅通过信号受体(B细胞抗原受体)捕获抗原，然后激活B细胞，而且它们还拥有TLRs，其参与本身有助于B细胞的激活。其他需要TLRs充分激活、产生细胞因子和展示共刺激分子的抗原提呈细胞通过其他方式捕获抗原，包括吞噬和受体介导的内吞作用，使用与B细胞不同的信号机制。因此，控制内体TLR运输和功能的细胞自主和特定细胞类型的因素可能是重要的。为了解决这些问题，产生了新的动物模型。其中包括一只TLR9-GFP转基因小鼠，并辅之以一只TLR7-GFP小鼠，以及一个通过体细胞核移植建立的跨核(TN)小鼠模型，该模型使用卵白蛋白特异性B细胞的核作为核供体；由此产生的小鼠拥有产生卵白蛋白特异性IgG1的B细胞。已经确定了Unc93B1在护送功能性内体TLRs到其作用部位的重要作用，但构成Unc93B1功能的许多生化和细胞生物学细节仍有待确定。TLR7和TLR9对Unc93B1内不同结构元素的不同要求需要分子解释。总而言之，用全新的小鼠模型进行的拟议实验的结果，应该可以阐明一类TLRs的细胞生物学和功能，这些TLRs对宿主防御具有已知的重要性，并与自身免疫有关。结合活细胞成像、生物化学和功能读数(细胞因子的产生、激活标记的表达、抗原呈递)将被应用于以下目的：1：TLR-GFP动物模型将用于研究内体Toll样受体7和9(TLR7/9)的配基结合、运输和蛋白水解性切割，以确定运输、信号和/或切割所需的新的TLR7/9辅助因子，并确定TLR7/9信号中的新成员。目的：将TLR-GFP小鼠与卵清蛋白特异性TN小鼠相结合，分析TLR和BCR信号在BCR和TLR配体结合反应中对B细胞活化的协同作用。目的3：利用新的化学酶工具研究TLRs的运输、相互作用和激活所需的UNC93B1的分子决定因素，以研究其拓扑结构和相互作用伙伴。

DESCRIPTION (provided by applicant): A detailed understanding of the trafficking behavior of the endosomal TLRs, as exemplified by that of TLR9, should aid in the design of strategies that improve delivery of antigen to processing compartments in professional antigen presenting cells, concomitant with appropriate activation of the antigen presenting cell. Consequently, this knowledge is expected to contribute to an understanding of the efficacy of vaccines currently in use, and how to improve on them. B cells represent a special target in this regard, as they not only capture antigen via a signaling receptor, the B cell receptor for antigen, which then activates the B cell, but they also possess TLRs whose engagement contributes to B cell activation per se. Other antigen presenting cells that require engagement of TLRs for full activation, production of cytokines and display of costimulatory molecules capture antigen by other means, including phagocytosis and receptor mediated endocytosis, using signaling apparatus distinct form that available to B cells. Cell-autonomous and cell type-specific factors that control endosomal TLR trafficking and function are therefore likely to be important. To address these questions, new animal models have been generated. These include a TLR9-GFP transgenic mouse, to be complemented by a TLR7-GFP mouse, and a transnuclear (TN) mouse model made by somatic cell nuclear transfer, using the nucleus of an ovalbumin-specific B cell as nucleus donor; the resulting mice possess B cells that produce ovalbumin-specific IgG1. Having established an important role for Unc93B1 in escorting functional endosomal TLRs to their site of action, much of the biochemical and cell biological details that underlie Unc93B1s function remain to be determined. The differential requirements displayed by TLR7 and TLR9 for distinct structural elements within Unc93B1 require a molecular explanation. Combined, the results of the proposed experiments, performed with altogether new mouse models, should illuminate the cell biology and function of a class of TLRs of known importance to host defense and implicated in autoimmunity. A combination of live cell imaging, biochemistry and functional readouts (cytokine production, expression of activation markers, antigen presentation) will be applied to the following aims: Aim 1: The TLR-GFP animal models will be applied to study endosomal Toll-like receptor 7 and 9 (TLR7/9) ligand binding, trafficking and proteolytic cleavage, to identify novel TLR7/9 cofactors required for trafficking, signaling and/or cleavage and to identify new players in TLR7/9 signaling. Aim 2: The TLR-GFP mice will be combined with the ovalbumin-specific TN mice to analyze the synergy between TLR and BCR signaling in B cell activation in response to a combination of BCR and TLR ligands. Aim 3: The molecular determinants of UNC93B1 required for trafficking, interaction and activation of TLRs will be examined, making use of new chemoenzymatic tools to study its topology and interacting partners.