Toll-like receptor 9: trafficking and signaling

Toll 样受体 9：运输和信号转导

• 批准号: 7922302
• 负责人: CYNTHIA A LEIFER
• 金额: $ 10.4万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-17 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7922302
• 关键词: Accounting; Address; Adjuvant; Affect; Allergic; Autoimmune Diseases; Autoimmune Process; Autoimmunity; B-Lymphocytes; Biological Assay; Bypass; Carbohydrates; Cell Fractionation; Cells; Communicable Diseases; Cytoplasmic Tail; DNA; Data; Dendritic Cells; Development; Discrimination; Disease; Dominant-Negative Mutation; Endocytosis; Endoplasmic Reticulum; Endosomes; Event; Goals; Golgi Apparatus; Immune response; Immune system; In Vitro; Interphase Cell; Knowledge; Lysine; Lysosomes; Malignant Neoplasms; Mass Spectrum Analysis; Methods; Modeling; Modification; Molecular; Movement; Mutate; Nucleic Acids; Pathology; Pathway interactions; Pattern; Peptide Hydrolases; Phosphorylation; Play; Post-Translational Protein Processing; Proteins; Receptors, Antigen, B-Cell; Regulation; Role; Secretory Cell; Signal Transduction; Site; Small Interfering RNA; Systemic Lupus Erythematosus; TLR9 gene; Testing; Therapeutic; Tyrosine; Ubiquitin; Ubiquitination; Vaccines; base; microbial; microorganism; mutant; novel; public health relevance; receptor; response; synergism; trafficking

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Despite incredible potential of CpG DNA to augment immune responses in cancer and infectious disease, inappropriate response to self DNA results in autoimmunity. This project seeks to identify key factors controlling localization of CpG DNA's specific receptor, Toll-like receptor 9. Through manipulating localization of TLR9, and thereby modulating CpG DNA activity, we hope to enhance vaccines and interrupt the cycle of autoimmune disease.

描述(申请人提供)：含有CpG基序的DNA(CpG DNA)在治疗癌症、传染病和过敏性疾病方面具有惊人的潜力。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，会引发系统性红斑狼疮等自身免疫性疾病。特异性受体TLR9的定位和转运可能在DNA自体/外源DNA识别中起关键作用。揭示TLR9转运的分子机制是我们朝着操纵TLR9转运以实现调控CpG DNA反应的长期目标迈出的第一步。我们已经证明，在CpG DNA刺激之前，TLR9定位于细胞内，主要分布在内质网(ER)。TLR9从内质网运输到内体和溶酶体，在那里它与内吞的CpG DNA共定位。这些数据提出了基本的问题，即是什么调节了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转运分析，我们已经积累了证据，证明TLR9结构性地退出内质网，CpG DNA诱导第二次TLR9转运事件，并且这两种事件都通过传统的细胞转运途径发生。因此，我们假设TLR9通过从高尔基复合体到内溶酶体的细胞分泌途径以高度调控的方式进行运输，高尔基转运是TLR9对CpG DNA反应的先决条件。我们认为，TLR9的转运可能与其他TLRs和自身免疫B细胞受体具有协同作用，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。首先，通过高尔基复合体的转基因和内源性TLR9运输将使用多种体外方法进行检测，包括一种新的蛋白酶裂解试验。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转运和TLR-自身免疫B细胞受体协同作用中的作用。通过了解TLR9贩运的机制，我们可以揭示对外来DNA的反应和对自身DNA缺乏反应的调节是如何实现的。这一认识将为CpG DNA作为佐剂和治疗剂的进一步开发提供基础，并开发中断自身免疫病理循环的机制。公共卫生相关性：尽管CpG DNA在癌症和传染病中具有增强免疫反应的惊人潜力，但对自身DNA的不当反应会导致自身免疫。本项目旨在确定控制CpG DNA特异性受体Toll样受体9定位的关键因素。通过操纵TLR9的定位，从而调节CpG DNA的活性，我们希望增强疫苗，中断自身免疫性疾病的循环。