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Molecular Biology of the Ribonuclease A Gene Superfamily

核糖核酸酶 A 基因超家族的分子生物学

基本信息

批准号：
7732597
负责人：
HELENE ROSENBERG
金额：
$ 68.17万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
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/7732597
关键词：
Ablation Anti-Bacterial Agents Antibodies Antigens Antiviral Agents Base Pairing Binding Sites Biochemical Biochemistry Biological Biology Butyric Acid Butyric Acids Cells Chemistry Chemotactic Factors Chickens Consensus Cytoplasmic Granules Data Endoribonucleases Enzymes Eosinophil cationic protein Eosinophil-Derived Neurotoxin Evolution Family Future GATA2 transcription factor Genes Genetic Genetic Transcription HL60 Host Defense Human Human Cloning Immune response Immune system Immunoglobulins Interleukin-10 Interleukin-13 Interleukin-5 Interleukin-6 Journals Laboratories Legal patent Leukocytes Ligands Manuscripts Molecular Molecular Biology Mus Myelogenous Nature Ovalbumin Pancreatic ribonuclease Peer Review Play Primates Production Progranulocytes Property RNA Interference RNase 2 Recording of previous events Reporter Reporting Ribonucleases Rodent Role Signal Pathway Site Structure TLR1 gene TLR2 gene TLR6 gene Therapeutic Agents Toll-Like Receptor 2 Transcriptional Regulation Work base chromatin immunoprecipitation editorial eosinophil human GATA1 protein promoter response secretory protein

项目摘要

During this reporting period, I joined with collaborators at NCI Frederick and reported that the eosinophil-derived neurotoxin (EDN) an eosinophil granule-derived secretory protein with ribonuclease and antiviral activity, activated myeloid DCs by triggering the Toll-like receptor (TLR)2-myeloid differentiation factor 88 signaling pathway, thus establishing EDN as an endogenous ligand of TLR2. EDN activates TLR2 independently of TLR1 or TLR6. When mice were immunized with ovalbumin (OVA) together with EDN or with EDN-treated OVA-loaded DCs, EDN enhanced OVA-specific T helper (Th)2-biased immune responses as indicated by predominant production of OVA-specific interleukin (IL)-5, IL-6, IL-10, and IL-13, as well as higher levels of immunoglobulin (Ig)G1 than IgG2a. Based on its ability to serve as a chemoattractant and activator of DCs, as well as the capacity to enhance antigen-specific immune responses, we consider EDN to have the properties of an endogenous alarmin that alerts the adaptive immune system for preferential enhancement of antigen-specific Th2 immune responses (J Exp Med, 2008). We have also continued our work on the transcriptional regulation of the genes encoding human EDN and ECP. Augmented expression of both GATA-1 and GATA-2 is detected in eosinophil promyelocyte HL-60 clone 15 cells in response to biochemical differentiation with butyric acid. Ablation of one or both consensus GATA binding sites in the extended 1000 base pair (bp) 5 promoter of EDN results in a profound reduction in reporter activity, while ablation of similar consensus GATA sites in the 5 promoter of ECP has no effect. Antibody-augmented electrophoretic mobility shift and chromatin immunoprecipitation analyses indicated that GATA-1 and GATA-2 proteins bind to the functional GATA sequences in the EDN promoter. Furthermore, although RNA silencing of GATA-1 alone had no impact on EDN expression, silencing of GATA-2 resulted in reduced expression of EDN and of GATA-1. Taken together, our data indicate that GATA-2 plays a major role in promoting transcription of EDN, both directly via interactions with the promoter and indirectly, via its ability to regulate expression of GATA-1 (Qiu et al., manuscript in review). I have also authored two invited, peer-reviewed manuscripts covering the field of RNase A biology, one entitled "RNase A ribonucleases and host defense: an evolving story" (J. Leukoc. Biol. 2008; 83: 1079-1087) and "Eosinophil-derived neurotoxin / RNase 2: connecting the past, the present and the future" (Current Pharm Biotechnol 2008; 9: 135-140). I have also been invited to join the Editorial Board of Journal of Biological Chemistry (July 2008 - 2013) largely due to expertise on ribonuclease chemistry and biochemistry.

在本报告所述期间，我与NCI Frederick的合作者一起报道，嗜酸性粒细胞衍生神经毒素(EDN)是一种嗜酸性粒细胞衍生的分泌蛋白，具有核糖核酸酶和抗病毒活性，通过触发Toll样受体(TLR)2-髓系分化因子88信号通路激活髓系树突状细胞，从而确立EDN作为TLR2的内源性配体。EDN独立于TLR1或TLR6激活TLR2。当卵蛋白(OVA)与EDN或EDN处理的OVA负载的DC一起免疫小鼠时，EDN增强了OVA特异性T辅助细胞(Th)2偏向的免疫反应，这表明主要产生OVA特异的IL-5、IL-6、IL-10和IL-13，以及比IgG2a更高水平的免疫球蛋白(Ig)G1。基于EDN作为DC的趋化剂和激活剂的能力，以及增强抗原特异性免疫反应的能力，我们认为EDN具有内源性警报蛋白的特性，可以提醒适应性免疫系统优先增强抗原特异性Th2免疫反应(J Exp Med，2008)。我们还继续研究人类EDN和ECP编码基因的转录调控。在嗜酸性粒细胞HL-60克隆15细胞中检测到GATA-1和GATA-2的表达增强，这是对丁酸的生化分化的反应。切割EDN延伸的1000个碱基对(BP)5启动子中的一个或两个共识GATA结合位点会导致报告活性显著降低，而切割ECP的5个启动子中类似的共识GATA位点则没有影响。抗体增强的电泳迁移率改变和染色质免疫沉淀分析表明，GATA-1和GATA-2蛋白与EDN启动子中的功能GATA序列结合。此外，尽管GATA-1的RNA沉默对EDN的表达没有影响，但GATA-2的沉默导致EDN和GATA-1的表达减少。综上所述，我们的数据表明，GATA-2直接通过与启动子的相互作用促进EDN的转录，也通过其调节GATA-1表达的能力间接地促进EDN的转录(邱等人，手稿在审查中)。我还写了两篇受邀的同行评议手稿，涵盖了RNaseA生物学领域，其中一篇题为“RNase A核糖核酸酶与宿主防御：一个进化的故事”(J.Leukoc)。比奥尔。2008年；83：1079-1087)和“嗜酸性粒细胞衍生神经毒素/核糖核酸酶2：连接过去、现在和未来”(现医药生物技术杂志2008；9：135-140)。我还被邀请加入《生物化学杂志》(2008年7月至2013年)的编辑委员会，这主要是因为我在核糖核酸酶化学和生物化学方面的专业知识。