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Understanding the early and late endosomal TLR9-mediated responses to viral DNA.

了解早期和晚期内体 TLR9 介导的病毒 DNA 反应。

基本信息

批准号：
8451259
负责人：
Yong-Jun Liu
金额：
$ 36.85万
依托单位：
BAYLOR RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-04-01 至 2017-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8451259
关键词：
Acids Affect Agonist Autoimmune Diseases Binding Proteins Biochemical Cell Line Cell Nucleus Cells Communicable Diseases Complex DNA Dendritic Cells Early Endosome Endoplasmic Reticulum Endosomes Exposure to Family Gene Expression Microarray Analysis Gene Targeting Genes Genetic Genomics Human IRAK1 gene IRAK4 gene Image Immune Immune system Immunologic Receptors Interferons Interleukin-6 Knock-out Libraries Location MAP3K7 gene Maps Mass Spectrum Analysis Mediating Methods Molecular Mus NF-kappa B Neurons Nucleic Acids Pathway interactions Play Production Protein Kinase C Proteins RNA Recruitment Activity Role Signal Transduction Small Interfering RNA TLR7 gene TNF gene TNF receptor-associated factor 3 TRAF4 gene TRAF6 gene Technology Viral Virus Virus Diseases Yeasts base casein kinase cytokine late endosome member microbial protein protein interaction research study response screening small hairpin RNA therapy development trafficking viral DNA viral RNA yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): Plasmacytoid dendritic cells (pDCs) are professional interferon (IFN)-producing cells of the immune system3-8 that are specialized in recognizing viral RNA and DNA via endosomal TLR7 and TLR9, respectively. Among the TLRs that associate with MyD88, TLR7 and TLR9 strictly depend on MyD88 for signal transduction, and reside in the endoplasmic reticulum (ER) in association with UNC93B and gp96. Engagement of TLR9 by CpG-A oligodeoxynucleotide (ODN) in the early endosomes preferentially triggers the TRAF3/IRAK1/IKK1/PI3K/IRF7 signal cascade leading to type 1 IFN responses, whereas engagement of TLR9 by CpG-B ODN in the late endosomes preferentially triggers the TRAF6/BTK/IRAK4/TAK1/IRF5/NF-kB signal cascade leading to the production of proinflammatory cytokines TNF and IL-6. However, the molecular mechanisms underlying the specialization of TLR9-mediated early endosomal IFN responses versus late endosomal proinflammatory cytokine responses are unknown. We discovered that PACSIN1, a member of the PACSIN (protein kinase C and casein kinase substrate in neurons) family, was specifically expressed by pDCs within the immune system. Our preliminary studies showed that knockdown of PACSIN1 expression in human pDCs or knockout of the Pacsin1 gene in mouse pDCs led to a great reduction in the early endosomal IFN response to CpG-A ODN without affecting the late endosomal proinflammatory cytokine response to CpG-B ODN. Our central hypothesis is that the molecular basis for the differential TLR9-mediated early and late endosomal responses to DNA is determined by the use of different adaptor molecules. The first aim will focus on the potential role of in CpG ODN trafficking by using a human pDC cell line with stable knockdown of PACSIN1 expression by shRNA and mouse pDCs derived from PACSIN-deficient mice. The second will focus on identification of PACSIN1-binding proteins by yeast two-hybrid screening and co-IP followed by mass spectrometry. The third aim will focus on further characterization of three potential PACSIN1-interacting molecules including CD2AP, MEKK4 and TRAF4.

描述（由申请人提供）：浆细胞样树突状细胞（pDC）是免疫系统中产生干扰素（IFN）的专业细胞3-8，专门分别通过内体TLR7和TLR9识别病毒RNA和DNA。在与MyD88相关的TLR中，TLR7和TLR9严格依赖MyD88进行信号转导，并与UNC93B和gp96相关地驻留在内质网(ER)中。早期内涵体中 CpG-A 寡脱氧核苷酸 (ODN) 与 TLR9 的结合优先触发 TRAF3/IRAK1/IKK1/PI3K/IRF7 信号级联，导致 1 型 IFN 应答，而晚期内涵体中 CpG-B ODN 与 TLR9 的结合优先触发 TRAF6/BTK/IRAK4/TAK1/IRF5/NF-kB 信号级联导致促炎细胞因子 TNF 和 IL-6 的产生。然而，TLR9 介导的早期内体 IFN 反应与晚期内体促炎细胞因子反应的特殊化背后的分子机制尚不清楚。我们发现 PACSIN1 是 PACSIN（神经元中的蛋白激酶 C 和酪蛋白激酶底物）家族的成员，由免疫系统内的 pDC 特异性表达。我们的初步研究表明，敲低人pDC中的PACSIN1表达或敲除小鼠pDC中的Pacsin1基因会导致早期内体IFN对CpG-A ODN的反应大大减少，而不影响晚期内体促炎细胞因子对CpG-B ODN的反应。我们的中心假设是，TLR9 介导的早期和晚期内体对 DNA 的差异性反应的分子基础是通过使用不同的接头分子来确定的。第一个目标将通过使用 shRNA 稳定敲低 PACSIN1 表达的人 pDC 细胞系和源自 PACSIN 缺陷小鼠的小鼠 pDC，重点关注 CpG ODN 运输中的潜在作用。第二个项目将重点通过酵母双杂交筛选和共免疫沉淀以及质谱分析来鉴定 PACSIN1 结合蛋白。第三个目标将集中于进一步表征三种潜在的 PACSIN1 相互作用分子，包括 CD2AP、MEKK4 和 TRAF4。