Gene Regulation In The Immune System

免疫系统中的基因调控

基本信息

批准号：
8553874
负责人：
Keiko Ozato
金额：
$ 85.78万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8553874
关键词：
Amino Acid Sequence Antibodies Autophagocytosis Autophagosome Binding Binding Proteins Cells ChIP-on-chip Chromatin Coupled Data Dendritic Cells Development Environment Gene Expression Regulation Gene Silencing Genes Genetic Transcription Goals Host Defense Human IRF3 gene Immune response Immune system Immunoblot Analysis Inflammatory Inflammatory Response Interferon Type II Interferons Interleukin-1 Interleukin-6 Ligands Lysosomes Mediating Modification Molecular Mycobacterium tuberculosis Natural Immunity Nuclear Nuclear Proteins Poly I-C Population Process Production Promoter Regions Proteins Relative (related person)Reporting Repression Resistance Role Signal Transduction Stimulus Testing Toll-Like Receptor Pathway Toll-like receptors Tuberculosis Tumor Necrosis Factor-alpha Ubiquitin Ubiquitin Like Proteins Ubiquitination Viral Yeasts antimicrobial cytokine follow-up gene repression genetic analysis human TNF protein interest link protein macrophage member pathogen protein degradation response transcription factor

项目摘要

Autophagy involves a long process that starts with signaling, followed by LC3 protein lipidation and autophagosome formation, leading to fusion with lysosomes and ubiquitin mediated protein degradation. Each of the above processes is controlled by autophagy specific genes conserved from yeast to humans. Our initial microarray and ChIP-on chip analyses indicated that some of autophagy related genes are downregulated in IRF8-/- DCs relative to IRF8+/+ DC. The follow-up study confirmed that IRF8 is required for expression of a large number of autophagy specific genes in both macrophages and DC. Some of these genes are induced by IFN and IRF8 bound to the upstream promoter regions. Consistent with these findings, IRF8 -/- macrophages were defective in lipidation mediated conversion of LC3 and nutophagosome formation. These results raise the possibility that IRF8 utilizes autophagy to eliminate certain pathogens including TB. Because IRF8 and several other IRF members are modified by both ubiquitin and SUMO, we asked whether these modifications occur in other proteins during innate immune responses. Macrophages were stimulated with IFN and a toll like receptor (TLR) ligand, CpG which activates the TLR pathway and triggers transcription of many genes important for macrophage function. Some of these genes are responsible for resistance against pathogens and other genes are involved in inflammatory responses in the host. To detect global ubiquitin or SUMO modifications of nuclear proteins in stimulated macrophages, we performed immunoblot analysis using antibody for ubiquitin, SUMO1 or SUMO2/3. To this end we tested nuclear fractions obtained from stimulated RAW264.7 macrophage cells. While there is only one ubiquitin protein, there are three SUMO molecules. SUMO1 is distinct from SUMO2 and SUMO3 that are more than 90% identical in amino acid sequence, and currently there is no antibody that distinguishes SUMO2 and SUMO3. We found that ubiquitin linked proteins were dramatically increased following IFNCpG stimulation. IFN treatment alone resulted in significant increase in ubiquitin-liked proteins. However, additional stimulationby CpG further increased the amount of ubiquitin bound proteins. Not only CpG, but other TLR ligands tested including LPS and poly IC also caused increased ubiquitination, provided that macrophages were prestimulated with IFN Similarly, IFN/TLR stimulation caused a marked increase in proteins conjugated to SUMO1 or SUMO2/3. Our data demonstrate that ubiquitin and SUMO modifications are integral part of innate immune responses, likely coupled with large changes in transcription and other nuclear activities, which helps accommodate a rapid response to pathogen stimuli. Transcriptionally active genes are embedded in chromatin that is dynamically exchanged, whereas silenced genes are surrounded by more stable chromatin. Chromatin environment

自噬涉及一个较长的过程，该过程始于信号传导，其次是LC3蛋白脂质和自噬体形成，从而导致与溶酶体和泛素介导的蛋白质降解。以上每个过程都由自噬特定基因从酵母到人类控制。我们最初的微阵列和芯片芯片分析表明，与IRF8+/+ DC相对于IRF8 - / - DC中的某些自噬相关基因被下调。后续研究证实，在巨噬细胞和DC中表达大量自噬特异性基因所必需的IRF8。这些基因中的一些是由IFN和IRF8与上游启动子区域结合的。与这些发现一致，IRF8 - / - 巨噬细胞在脂质中介导的LC3和果蝇形成的转化率有缺陷。这些结果增加了IRF8利用自噬来消除包括结核病在内的某些病原体的可能性。由于IRF8和其他几个IRF成员都被泛素和相扑都改变了，因此我们询问在先天免疫反应期间其他蛋白质中是否发生了这些修饰。用IFN刺激巨噬细胞，并像受体（TLR）配体一样刺激巨噬细胞，CPG激活TLR途径并触发许多对巨噬细胞功能重要的基因的转录。其中一些基因负责对病原体的抗性，而其他基因参与宿主中的炎症反应。为了检测刺激巨噬细胞中核蛋白的全球泛素或相扑修饰，我们使用泛素，SUMO1或SUMO2/3进行了免疫印迹分析。为此，我们测试了从刺激的RAW264.7巨噬细胞中获得的核部分。虽然只有一种泛素蛋白，但共有三个分子。 SUMO1与SUMO2和SUMO3不同，在氨基酸序列中的90％以上，目前没有区分SUMO2和SUMO3的抗体。我们发现，IFNCPG刺激后，泛素连接的蛋白质大大增加。仅IFN治疗会导致泛素蛋白质的显着增加。但是，通过CpG进行的额外刺激进一步增加了泛素结合蛋白的量。不仅CPG，而且包括LPS和Poly IC在内的其他TLR配体也导致了泛素化的增加，只要巨噬细胞与IFN相似，IFN/TLR刺激会导致与SUMO1或SUMO2或SUMO2或SUMO2或3的IFN/TLR刺激导致明显的增加。我们的数据表明，泛素和相扑修饰是先天免疫反应不可或缺的一部分，可能加上转录和其他核活动的巨大变化，这有助于适应对病原体刺激的快速反应。转录活性基因嵌入了动态交换的染色质中，而沉默的基因被更稳定的染色质包围。染色质环境