Molecular Basis for PKR Activation by Viral RNAs

病毒 RNA 激活 PKR 的分子基础

• 批准号: 7132072
• 负责人: JOSEPH D PUGLISI
• 金额: $ 29.64万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7132072
• 关键词: X ray crystallography; adenosine triphosphate; calorimetry; chemical kinetics; conformation; double stranded RNA; enzyme activity; enzyme inhibitors; enzyme mechanism; fluorescence spectrometry; genetic regulation; genetic translation; immune response; intermolecular interaction; nanotechnology; nuclear magnetic resonance spectroscopy; phosphorylation; protein binding; protein kinase; protein structure function; thermodynamics; virus RNA; virus diseases

DESCRIPTION (provided by applicant): Translation is highly regulated. Phosphorylation of translation initiation factors has been shown to be a key feature of regulation in eukaryotes. Double-stranded RNA dependent protein kinase (PKR) is a 68 kDa protein that is activated by dsRNA binding, leading to phosphorylation of initiation factor elF2a. Phosphorylation of elF2 leads to blockage of nucleotide exchange by elF2B, resulting in down regulation of translation. PKR function has been linked to innate immune antiviral response, and its activity is modulated in many cancers. Viral RNAs can activate PKR, and several viruses encode RNAs and proteins that inhibit PKR function. Despite a decade of biochemical and structural studies, the basis for RNA activation of PKR is not known. Here we propose to study the linkage between double-stranded RNA binding and kinase activation. In specific aim 1, we will define a set of RNA activators and inhibitors of PKR that bind specifically to the tandem double-stranded RNA binding motifs (dsRBD1,2) of PKR. We will determine the thermodynamic parameters that govern RNA-protein recognition, and establish defined systems for structural studies. In specific aim 2, the structures of dsRBD1,2 in complex with several of these viral RNAs-HlV TAR, Epstein Barr virus EBER and adenovirus VAI, will be determined by NMR to establish the structural basis for RNA-protein recognition. In specific aim 3, the mechanism of PKR will be investigated using a variety of biochemical and biophysical methods. We will delineate the kinetic framework for kinase function, and compare the action of RNA activators and inhibitors. In specific aim 4, we will study the structural features and conformational dynamics of full-length PKR using NMR spectroscopy and single-molecule fluorescence approaches. We will attempt to link the kinetic observations of aim 3 with changes in PKR conformation and aggregation state as a function of RNA binding and autophosphorylation. Finally in specific aim 5, we will link the molecular mechanism for RNA activation of PKR with the effects of PKR on translation in extracts. The proposal links basic physical principles of RNA-protein recognition with a molecular view of PKR activation and effects on translation. The results of this work will explain a central mechanism of gene regulation required for antiviral response and control of cell proliferation.

描述（由申请人提供）：翻译受到严格监管。翻译起始因子的磷酸化已被证明是真核生物调节的关键特征。双链 RNA 依赖性蛋白激酶 (PKR) 是一种 68 kDa 的蛋白质，通过 dsRNA 结合激活，导致起始因子 eIF2a 磷酸化。 eF2 的磷酸化会导致 eF2B 阻断核苷酸交换，从而导致翻译下调。 PKR 功能与先天免疫抗病毒反应有关，其活性在许多癌症中受到调节。病毒RNA可以激活PKR，并且一些病毒编码抑制PKR功能的RNA和蛋白质。尽管进行了十年的生化和结构研究，但 PKR RNA 激活的基础尚不清楚。在这里，我们建议研究双链 RNA 结合和激酶激活之间的联系。在具体目标 1 中，我们将定义一组 PKR 的 RNA 激活剂和抑制剂，它们特异性结合 PKR 的串联双链 RNA 结合基序 (dsRBD1,2)。我们将确定控制 RNA 蛋白质识别的热力学参数，并建立用于结构研究的明确系统。在具体目标2中，将通过NMR测定与这些病毒RNA-HIV TAR、EB病毒EBER和腺病毒VAI中的几种复合物的dsRBD1,2的结构，以建立RNA-蛋白质识别的结构基础。在具体目标 3 中，将使用各种生化和生物物理方法研究 PKR 的机制。我们将描绘激酶功能的动力学框架，并比较 RNA 激活剂和抑制剂的作用。在具体目标 4 中，我们将使用核磁共振波谱和单分子荧光方法研究全长 PKR 的结构特征和构象动力学。我们将尝试将目标 3 的动力学观察与 PKR 构象和聚集状态的变化联系起来，作为 RNA 结合和自磷酸化的函数。最后，在具体目标 5 中，我们将 PKR RNA 激活的分子机制与 PKR 对提取物翻译的影响联系起来。该提案将 RNA 蛋白质识别的基本物理原理与 PKR 激活和翻译影响的分子观点联系起来。这项工作的结果将解释抗病毒反应和细胞增殖控制所需的基因调控的中心机制。