Endosomal TLRs and their accessory proteins: cell biology and biochemistry

描述（由申请人提供）：详细了解内体TLR的运输行为，如TLR 9的运输行为所示，应有助于设计改善抗原递送至专职抗原呈递细胞中的加工区室的策略，同时适当激活抗原呈递细胞。因此，这些知识有望有助于了解目前使用的疫苗的效力，以及如何改进这些疫苗。在这方面，B细胞代表了特殊的靶标，因为它们不仅通过信号传导受体（抗原的B细胞受体）捕获抗原，然后激活B细胞，而且它们还具有TLR，其接合有助于B细胞本身的激活。需要TLR参与以完全活化、产生细胞因子和展示共刺激分子的其他抗原呈递细胞通过其他方式捕获抗原，包括吞噬作用和受体介导的内吞作用，使用B细胞可获得的不同形式的信号传导装置。因此，控制内体TLR运输和功能的细胞自主和细胞类型特异性因子可能是重要的。 为了解决这些问题，已经产生了新的动物模型。这些包括TLR 9-GFP转基因小鼠，由TLR 7-GFP小鼠补充，和通过体细胞核移植制备的跨核（TN）小鼠模型，使用卵清蛋白特异性B细胞的细胞核作为细胞核供体;所得小鼠具有产生卵清蛋白特异性IgG 1的B细胞。Unc 93 B1在护送功能性内体TLR至其作用位点中具有重要作用，但Unc 93 B1功能的许多生化和细胞生物学细节仍有待确定。TLR 7和TLR 9对Unc 93 B1内不同结构元件的不同要求需要分子解释。结合起来，用全新的小鼠模型进行的拟议实验的结果应该阐明一类TLR的细胞生物学和功能，这些TLR对宿主防御具有已知的重要性，并与自身免疫有关。活细胞成像、生物化学和功能读数的组合（细胞因子的产生、活化标志物的表达、抗原呈递）将应用于以下目的：TLR-GFP动物模型将用于研究内体Toll样受体7和9（TLR 7/9）配体结合、运输和蛋白水解切割，以鉴定运输所需的新型TLR 7/9辅因子，本发明的目的在于鉴定TLR 7/9信号传导和/或切割中的新参与者。目标二：将TLR-GFP小鼠与卵清蛋白特异性TN小鼠组合，以分析响应于BCR和TLR配体的组合的B细胞活化中TLR和BCR信号传导之间的协同作用。目标3：UNC 93 B1的分子决定因素所需的TLRs的运输，相互作用和激活将进行检查，利用新的化学酶的工具来研究其拓扑结构和相互作用的合作伙伴。 公共卫生相关性：项目说明。通过构建GFP标记的TLRs转基因动物，提出了新的动物模型来研究内体TLRs（TLR 7和TLR 9）及其重要伴侣Unc 93 B1的运输特性和相互作用蛋白。这些模型将与通过体细胞核移植产生的具有卵清蛋白特异性B细胞的动物的跨核小鼠组合，以使用活细胞成像和生物化学研究TLR和BCR衍生信号之间的相互作用，用于适当的B细胞活化。

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. PUBLIC HEALTH RELEVANCE: Project narrative. New animal models are proposed to study the trafficking properties and interacting proteins of the endosomal TLRs (TLR7 and TLR9) and their essential partner, Unc93B1, through construction of animals transgenic for GFP-tagged TLRs. These models will be combined with transnuclear mice, created by somatic cell nuclear transfer to yield animals with ovalbumin-specific B cells, to study the interplay between TLR- and BCR derived signals for proper B cell activation, using live cell imaging and biochemistry.