Toll-like receptor 9: trafficking and signaling

Toll 样受体 9：运输和信号转导

• 批准号: 7742607
• 负责人: CYNTHIA A LEIFER
• 金额: $ 34.3万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7742607
• 关键词: Accounting; Address; Adjuvant; Affect; Allergic Disease; Autoimmune Diseases; Autoimmune Process; Autoimmunity; B-Lymphocytes; Biological Assay; Bypass; Carbohydrates; Cell Fractionation; Cells; Communicable Diseases; Cytoplasmic Tail; DNA; Data; Dendritic Cells; Development; Discrimination; 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（由申请人提供）：含有CPG基序的DNA（CPG DNA）具有治疗癌症，传染性和过敏性疾病的难以置信的潜力。尽管有潜力，但对我们自己的核酸（包括DNA）的反应会触发自身免疫性疾病，例如全身性红斑狼疮。特定受体TLR9的本地化和运输可能在自我/外国DNA歧视中起关键作用。揭示TLR9运输的分子机制是朝着操纵TLR9运输以实现CPG DNA响应调节的长期目标的第一步。我们已经表明，在CpG DNA刺激之前，TLR9是细胞内的，主要是在内质网（ER）中。 TLR9从ER到内体和溶酶体的运输与内吞CPG DNA共定位。这些数据提出了哪些调节TLR9访问CPG DNA的基本问题，访问如何影响对自我和微生物DNA的反应？使用新的TLR9运输分析，我们积累了证据，表明TLR9组成型退出ER，CPG DNA诱导了次级TLR9运输事件，并且这两个事件都通过传统的细胞运输途径发生。因此，我们假设TLR9通过从高尔基体复合体到内溶性的细胞分泌途径以高度调节的方式组成运输，而高尔基传输是TLR9对CpG DNA响应的先决条件。我们认为，TLR9运输可能与其他TLR和自身免疫性B细胞受体有关，并受到TLR9细胞质尾巴的翻译后修饰的调节。该假设将以三个特定目的进行检验。首先，将使用多种体外方法（包括一种新型的蛋白酶裂解测定法）对通过高尔基体复合物进行转染和内源性TLR9运输。其次，我们将研究TLR9-Automune 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贩运和TLR-Automune B细胞受体协同中的作用。通过了解TLR9运输的机制，我们可以发现如何实现对外国DNA的反应和对自我DNA的反应的调节。这些知识将为CpG DNA作为辅助和治疗性的进一步发展提供基础，并开发出中断自身免疫性病理周期的机制。公共卫生相关性：尽管CPG DNA可能会增加癌症和传染病的免疫反应的潜力，但对自我DNA的反应不当会导致自身免疫性。该项目旨在确定控制CpG DNA特定受体的定位的关键因素，Toll样受体9。通过操纵TLR9的定位，从而调节CpG DNA活性，我们希望增强疫苗并中断自身免疫性疾病的循环。