Interferon Action and Protein Phosphorylation

干扰素作用和蛋白质磷酸化

基本信息

批准号：
7157561
负责人：
Charles E Samuel
金额：
$ 27.73万
依托单位：
UNIVERSITY OF CALIFORNIA SANTA BARBARA
依托单位国家：
美国
项目类别：
财政年份：
1983
资助国家：
美国
起止时间：
1983-12-01 至 2009-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7157561
关键词：
5&apos Flanking Region Adenoviruses Animals Binding Binding Sites Biochemical Biochemical Genetics Biological Assay Cell Line Cells Chemicals Chimeric Proteins Clinic Complex Conserved Sequence Cultured Cells Custom DNA DNA Binding DNA Microarray Chip DNA Microarray format DNase-I Footprinting Disruption Double-Stranded RNA Elements Endoribonucleases Event Gel Gene Expression Gene Targeting Genes Genetic Transcription Growth HIV Health Human Interferons Investigation Maps Mediating Molecular Molecular Genetics Mouse Cell Line Mouse Strains Mus Mutagenesis Normal Cell Nucleotides Numbers Pancreatic ribonuclease Phenotype Phosphorylation Phosphotransferases Positioning Attribute Primer Extension Promoter Regions Property Protein Binding Protein Biosynthesis Proteins RNA RNase protection assay Recombinant Interferon Regulation Relative (related person)Reovirus Reporter Research Response Elements Ribonucleases Roentgen Rays Role Site Structure Tacrolimus Binding Proteins Tars Tissues Transcript Transfection Viral Virus Virus Diseases Virus Replication Work aptamer base cytokine eIF-2 Kinase genetic regulatory protein indexing insight mutant novel nuclease promoter protein function research study response stem

项目摘要

The OVERALL OBJECTIVE of the proposed investigation is to elucidate the role of the interferon-inducible double-stranded RNA-dependent protein kinase (PKR) in the actions which natural and recombinant interferons mediate on viral and host functions. The SPECIFIC AIMS of our proposed continuation investigation of protein phosphorylation and interferon (IFN) action are as follows: (1). To further delineate the structure of the 5'-flanking region of the Pkr gene required for interferon-inducible as well as basal transcriptional activity. To map the major Pkr transcription sites, using RNA from tissues of uninfected and infected mice and from untreated and IFN-treated cells. To elucidate the importance of potential protein binding sites in Pkr promoter activity, with emphasis placed on proteins binding the novel 15 nucleotide DNA element designated KCS which so far is unique to the human and mouse Pkr promoters. To examine the ability ofcytokines and factors other than IFNs to modulate P_ expression. (2). To further characterize the biochemical and biophysical properties of the PKR kinase. To attempt to identif3' definitively the sites of PKR autophosphorylation associated with activation. To characterize the structural basis and functional significance of PKR protein interactions, and to continue efforts to obtain a crystal structure of PKR with the hope that the structure will provide further insight into function. (3). To characterize the expression and function ofwildtype and mutant PKR proteins in cell culture. To assess the effect of singular expression of PKR forms in transfected PKR-deficient cells. To use RNase protection and DNA microarray approaches to gain insights into role of PKR on transcript profiles in untreated, IFN-treated, and infected cells from two strains of mice. The health relatedness of the proposed research stems from the likelihood that the work may contribute to a better understanding of regulatory mechanisms operative in normal cells as well as virus-infected cells. Elucidation of the actions of IFN at the molecular level is of immediate importance in view of the use of IFN in the clinic.

提出的研究的总体目的是阐明干扰素诱导的作用双链RNA依赖性蛋白激酶（PKR）在天然和重组干扰素的作用中介导病毒和宿主功能。我们提出的蛋白质持续研究的具体目的磷酸化和干扰素（IFN）作用如下：（1）。进一步描述干扰素诱导所需的PKR基因的5'-倾斜区域的结构以及基础转录活性。绘制主要的PKR转录位点，使用来自未感染和感染的小鼠以及未处理和IFN处理的细胞。阐明潜在蛋白质的重要性 PKR启动子活性中的结合位点，重点放在结合新型蛋白质的15个核苷酸DNA元素上到目前为止，指定的KC是人类和小鼠PKR启动子独有的。检查循环因子和除IFN以外的其他因素调节P_表达。（2）。为了进一步表征PKR激酶的生化和生物物理特性。尝试确定与激活相关的PKR自磷酸化的位点。为了表征结构 PKR蛋白相互作用的基础和功能意义，并继续努力获得晶体结构 PKR希望该结构能为功能提供进一步的见解。（3）。表征WildType和突变体PKR蛋白在细胞培养中的表达和功能。到评估PKR形式在转染的PKR缺陷细胞中的奇异表达的影响。使用RNase保护和 DNA微阵列的方法可以深入了解PKR在未处理，IFN处理和来自两种小鼠菌株的感染细胞。拟议研究的健康相关性源于该工作可能有助于更好的可能性了解正常细胞和病毒感染细胞中操作的调节机制。阐明鉴于在诊所中使用IFN，IFN在分子水平上的作用至关重要。