Toll-like receptor 9: trafficking and signaling

摘要 含有CpG基序的DNA(CpG DNA)在治疗癌症、传染性和过敏性疾病方面具有惊人的潜力 疾病。尽管有这种潜力，但对包括DNA在内的我们自己的核酸的反应会触发自身免疫 系统性红斑狼疮等疾病。特异性受体TLR9的定位和运输， 可能在自/外DNA歧视中起关键作用。揭示TLR9的分子机制 人口贩运是我们实现操纵TLR9人口贩运的长期目标的第一步 CpG DNA反应的调控。我们已经证明TLR9定位于细胞内，主要分布在 内质网(ER)，在CpG DNA刺激之前。TLR9从内质网到内体和 与内吞的CpG DNA共定位的溶酶体。这些数据提出了一些根本性的问题 是什么调节TLR9对CpG DNA的访问，以及访问如何影响对自身和微生物DNA的反应？ 使用新的TLR9贩运分析，我们已经积累了TLR9构成地退出ER的证据，即 CpG DNA诱导TLR9二次转运事件，两种事件均通过传统细胞发生 贩运路径。因此，我们假设TLR9在高度集中的情况下 通过细胞分泌途径从高尔基复合体到 此外，高尔基体转运是TLR9对CpG DNA应答的先决条件。我们 相信TLR9的贩运可能是与其他TLR和自身免疫B细胞协同作用的原因 细胞受体，并受TLR9细胞质尾部的翻译后修饰调控。 这一假设将在三个具体目标上得到检验。第一，转基因和内源性TLR9的贩运 通过高尔基复合体将使用包括一种新的蛋白酶在内的多种体外方法进行检测 卵裂试验。其次，我们将研究TLR9-自身免疫B细胞受体协同作用的机制。 第三，我们将确定TLR9翻译后修饰在调节细胞内的作用 运输和TLR自身免疫B细胞受体的协同作用。通过了解TLR9的机制 贩运我们可以揭示对外来DNA的反应和对自身DNA的缺乏反应是如何调节的 已实现。这一认识将为进一步开发CpG DNA作为佐剂和 治疗以及开发机制，以中断自身免疫病理的循环。

ABSTRACT DNA containing CpG motifs (CpG DNA) has incredible potential to treat cancer, infectious and allergic diseases. Despite this potential, response to our own nucleic acids, including DNA, triggers autoimmune diseases such as systemic lupus erythematosus. Localization and trafficking of the specific receptor, TLR9, may play a key role in self/foreign DNA discrimination. Uncovering the molecular mechanisms of TLR9 trafficking is the first step towards our long term goal of manipulating TLR9 trafficking to achieve modulation of CpG DNA response. We have shown that TLR9 is localized intracellularly, predominantly in the endoplasmic reticulum (ER), prior to CpG DNA stimulation. TLR9 traffics from the ER to endosomes and lysosomes where it co-localizes with endocytosed CpG DNA. These data raise the fundamental questions of what regulates TLR9 access to CpG DNA and how does access affect response to self and microbial DNA? Using new TLR9 trafficking assays, we have accumulated evidence that TLR9 constitutively exits the ER, that CpG DNA induces a secondary TLR9 trafficking event, and that both events occur through traditional cell trafficking pathways. Therefore, we hypothesize that TLR9 constitutively traffics in a highly regulated fashion through the cell secretory pathway from the Golgi complex to endolysosomes and that Golgi transit is a prerequisite for TLR9 response to CpG DNA. We believe that TLR9 trafficking may account for synergy with other TLRs and the autoimmune B cell receptor, and is regulated by post-translational modification of the TLR9 cytoplasmic tail. This hypothesis will be tested in three Specific Aims. First, transfected and endogenous TLR9 trafficking through the Golgi complex will be examined using multiple in vitro approaches including a novel protease cleavage assay. Second, we will examine the mechanism of TLR9-autoimmune B cell receptor synergism. Third, we will determine the role of TLR9 post-translational modification in regulating intracellular trafficking and in TLR-autoimmune B cell receptor synergy. By understanding the mechanism of TLR9 trafficking we can uncover how regulation of response to foreign DNA and lack of response to self-DNA is achieved. This knowledge will provide the basis for the further development of CpG DNA as an adjuvant and therapeutic as well as develop mechanisms to interrupt the cycle of autoimmune pathology.