Endosomal TLRs and their accessory proteins: cell biology and biochemistry

描述（由申请人提供）：对内体tlr转运行为的详细了解，如TLR9的转运行为，应有助于设计策略，以改善抗原递送到专业抗原提呈细胞的加工区，同时适当激活抗原提呈细胞。因此，预计这些知识将有助于了解目前使用的疫苗的效力，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tlr，其参与有助于B细胞本身的激活。其他抗原呈递细胞需要tlr的参与才能完全激活，产生细胞因子和显示共刺激分子，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很重要。为了解决这些问题，新的动物模型已经产生。其中包括TLR9-GFP转基因小鼠，与TLR7-GFP小鼠互补，以及通过体细胞核转移制成的跨核（TN）小鼠模型，使用卵清蛋白特异性B细胞的细胞核作为核供体；由此产生的小鼠拥有产生卵清蛋白特异性IgG1的B细胞。在确定了Unc93B1在护送功能性内体tlr到达其作用位点方面的重要作用后，Unc93B1功能背后的许多生化和细胞生物学细节仍有待确定。TLR7和TLR9对Unc93B1中不同结构元素的不同需求需要分子解释。综上所述，采用全新的小鼠模型进行的实验结果，应该能够阐明一类已知对宿主防御和自身免疫具有重要意义的tlr的细胞生物学和功能。活细胞成像、生物化学和功能数据（细胞因子产生、激活标记物表达、抗原呈递）的结合将应用于以下目标：目标1:TLR-GFP动物模型将用于研究内体toll样受体7和9 （TLR7/9）配体结合、运输和蛋白水解裂解，以确定运输、信号传导和/或裂解所需的新型TLR7/9辅助因子，并确定TLR7/9信号传导的新参与者。目的2：将TLR- gfp小鼠与卵清蛋白特异性TN小鼠结合，分析在BCR和TLR配体联合作用下，TLR和BCR信号在B细胞活化中的协同作用。目标3：将研究tlr转运、相互作用和激活所需的UNC93B1分子决定因素，利用新的化学酶工具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.