BAF: an Intrinsic Host Defense Responsive to Foreign DNA

BAF：对外来 DNA 做出反应的内在宿主防御

基本信息

批准号：
8037692
负责人：
MATTHEW S WIEBE
金额：
$ 10.8万
依托单位：
UNIVERSITY OF NEBRASKA LINCOLN
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-03-04 至 2013-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8037692
关键词：
Autoimmune Diseases Binding Biological Assay Biosensor Cell Nucleus Cells Cellular biology Co-Immunoprecipitations Cytoplasm DNA DNA Binding DNA biosynthesis DNA-Binding Proteins Data Deoxyribonucleases Development Environment Eukaryotic Cell Event Funding Gene Expression Genes Genetic Materials Genetic Transcription Genome Goals Host Defense Immune Immune response Immune system Immunofluorescence Immunologic Infection Interferons Invaded Mass Spectrum Analysis Mediating Methods Molecular Monkeypox Mutation Nuclear Envelope Nuclear Import Nucleic Acids Pathway interactions Phase Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Plasmids Poxviridae Precipitation Property Protein Kinase Proteins Reaction Recruitment Activity Regulation Reporter Genes Repression Role Signal Pathway Signal Transduction Smallpox Source Specificity Staging Sum TLR9 gene Temperature Time Transfection Vaccination Vaccinia Vaccinium Virus barrier-to-autointegration factor base established cell line gene therapy inhibitor/antagonist mutant novel overexpression pathogen plasmid DNA response sensor viral DNA

项目摘要

DESCRIPTION (provided by applicant): Recognition of a pathogen's genetic material as "foreign" alerts the cell to the presence of an invader. At the same time, an unchecked immune reaction to host nucleic acid incorrectly targets healthy cells for destruction, leading to autoimmune disease. It is therefore critical to understand how our immune systems detect foreign DNA, as well as the control mechanisms that restrain them in the absence of a true threat. In this application, we demonstrate that the barrier to autointegration factor (BAF) is a novel DMA-specific host defense protein. We have identified BAF as an inhibitor of poxviral DNA replication and hypothesize that BAF responds to any cytoplasmic DNA. As outlined in our three Specific Aims, our primary goal is to examine how BAF contributes to the cellular recognition and response to foreign DNA. In the first aim, we will clarify our understanding of the mechanism(s) through which BAF can inhibit various stages of the poxviral lifecycle. Mutant forms of BAF, established cell lines that overexpress or have been depleted of BAF, and both wild-type vaccinia and temperature-sensitive viruses will be utilized. The second aim builds upon our recent observation that BAF relocalizes to discrete foci upon transfection of plasmid DNA, and retards expression of genes encoded by these plasmids, indicating that BAF targets multiple sources of DNA. We will employ a battery of approaches including immunofluorescence analysis, BAF:plasmid co- precipitation studies, and reporter gene assays to characterize the recruitment of BAF to foreign DNA and its inhibitory impact upon binding. The third aim will focus on two general themes: [1] understanding how BAF's ability to function as a DNA sensor is regulated by dynamic phosphorylation, and [2] determining whether BAF's repression of foreign DNA is accompanied by activation of, or intersection with, cellular signaling pathways. In sum, the studies outlined here will further our understanding of BAF as a novel host defense protein. Understanding and exploiting the host/pathogen interactions that accompany infection with variola, the etiological agent of smallpox and a potential bioterrorist threat, and monkeypox, an emerging pathogen, is of significant biomedical importance. Exploring BAF's response against foreign DNA will also illuminate events that occur during other types of infections or the development of autoimmune disease, and will be of relevance to the establishment of effective strategies for gene therapy and DNA vaccination.

描述（由申请人提供）：将病原体的遗传物质识别为“外来”会提醒细胞入侵者的存在。与此同时，对宿主核酸的未经控制的免疫反应错误地瞄准健康细胞进行破坏，导致自身免疫性疾病。因此，了解我们的免疫系统如何检测外来 DNA，以及在没有真正威胁的情况下抑制它们的控制机制至关重要。在此应用中，我们证明自动整合屏障因子 (BAF) 是一种新型 DMA 特异性宿主防御蛋白。我们已经确定 BAF 是痘病毒 DNA 复制的抑制剂，并假设 BAF 对任何细胞质 DNA 都有反应。正如我们的三个具体目标中所述，我们的主要目标是研究 BAF 如何促进细胞识别和对外源 DNA 做出反应。第一个目标是阐明我们对 BAF 抑制痘病毒生命周期各个阶段的机制的理解。将利用 BAF 的突变形式、过度表达或已耗尽 BAF 的已建立细胞系以及野生型牛痘和温度敏感病毒。第二个目标基于我们最近的观察，即 BAF 在转染质粒 DNA 后重新定位到离散的焦点，并延迟这些质粒编码的基因的表达，表明 BAF 靶向多种 DNA 来源。我们将采用一系列方法，包括免疫荧光分析、BAF：质粒共沉淀研究和报告基因测定，来表征 BAF 对外源 DNA 的募集及其对结合的抑制影响。第三个目标将集中于两个一般主题：[1]了解 BAF 作为 DNA 传感器的功能如何受到动态磷酸化的调节，以及 [2]确定 BAF 对外源 DNA 的抑制是否伴随着以下物质的激活或交叉：细胞信号传导途径。总之，这里概述的研究将进一步加深我们对 BAF 作为一种新型宿主防御蛋白的理解。了解和利用天花（天花的病原体和潜在的生物恐怖威胁）和猴痘（一种新出现的病原体）感染伴随的宿主/病原体相互作用具有重要的生物医学意义。探索 BAF 对外源 DNA 的反应也将阐明其他类型感染或自身免疫性疾病发展过程中发生的事件，并将与建立有效的基因治疗和 DNA 疫苗接种策略相关。