Structural studies of PKR regulation by viral non-coding RNA

病毒非编码RNA调控PKR的结构研究

基本信息

批准号：
8496700
负责人：
Graeme L Conn
金额：
$ 14.66万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-01 至 2014-12-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8496700
关键词：
Address Antiviral Response Apoptosis Back Bacterial Infections Binding Sites Biochemical Biochemistry Biological Assay Biology Calorimetry Cell physiology Cells Cellular Stress Cellular biology Chemistry Complex Crystallization Crystallography Development Diagnostic Double-Stranded RNA Drug Targeting Engineering Ensure Enzymes Foundations Functional RNA Future Gel Chromatography Gene Expression Generations Goals Growth Factor Heterogeneity Human Immune response Immune system Knowledge Laboratories Libraries Malignant Neoplasms Mammalian Cell Measurement Mediating Mediator of activation protein Metabolic Diseases Mitotic Cell Cycle Modeling Molecular Nerve Degeneration Neurodegenerative Disorders Obesity Outcome Phase Phosphotransferases Process Proteins RNA RNA Binding RNA Conformation RNA Folding RNA-Protein Interaction Race Reagent Regulation Research Research Personnel Resolution Roentgen Rays Role Selenomethionine Signal Transduction Structure Synchrotrons Technology Therapeutic Titrations Transcript U1A protein Variant Viral Virus Diseases X ray diffraction analysis X-Ray Diffraction arm cell growth deprivation design design and construction eIF-2 Kinase experience feeding human disease inhibitor/antagonist innovation insight interest melting novel novel strategies pathogen programs protein complex receptor research study response screening small molecule success therapeutic target viral RNA

Address Antiviral Response Apoptosis Back Bacterial Infections Binding Sites Biochemical Biochemistry Biological Assay Biology Calorimetry Cell physiology Cells Cellular Stress Cellular biology Chemistry Complex Crystallization Crystallography Development Diagnostic Double-Stranded RNA Drug Targeting Engineering Ensure Enzymes Foundations Functional RNA Future Gel Chromatography Gene Expression Generations Goals Growth Factor Heterogeneity Human Immune response Immune system Knowledge Laboratories Libraries Malignant Neoplasms Mammalian Cell Measurement Mediating Mediator of activation protein Metabolic Diseases Mitotic Cell Cycle Modeling Molecular Nerve Degeneration Neurodegenerative Disorders Obesity Outcome Phase Phosphotransferases Process Proteins RNA RNA Binding RNA Conformation RNA Folding RNA-Protein Interaction Race Reagent Regulation Research Research Personnel Resolution Roentgen Rays Role Selenomethionine Signal Transduction Structure Synchrotrons Technology Therapeutic Titrations Transcript U1A protein Variant Viral Virus Diseases X ray diffraction analysis X-Ray Diffraction arm cell growth deprivation design design and construction eIF-2 Kinase experience feeding human disease inhibitor/antagonist innovation insight interest melting novel novel strategies pathogen programs protein complex receptor research study response screening small molecule success therapeutic target viral RNA

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项目摘要

DESCRIPTION (provided by applicant): PKR is a key component of the cellular innate immune response against viral or bacterial infection and is an important mediator and integrator of signals that control other diverse cellular processes such as the cell cycle, cell growth, apoptosis, and the response to cell stresses such as growth factor deprivation. Aberrant PKR function is also associated with human diseases including cancers, neurodegenerative diseases and, potentially, metabolic disorders and obesity. Our understanding of PKR regulation by RNA, or other molecules, and its potential as a drug target are currently hampered by the lack of high-resolution structural information for a PKR-RNA complex. This proposal describes a research program to obtain crystals of a double-stranded RNA-activated protein kinase (PKR):non-coding viral RNA complex in order to produce the first high-resolution molecular snapshot of this important cellular kinase and its regulation by RNA. Our approaches to this major challenge are grouped into two integrated aims. First, since the most critical determinant of success in RNA crystallography is the RNA construct itself, Specific Aim 1 will exploit the detailed biochemical understanding we have developed of the non-coding adenoviral transcript VA RNAI to produce a diverse 'library' of novel RNA constructs for crystallization with PKR. These include systematic variation of the RNA structural domains, introduction of compact and stable RNA secondary structures such as tetraloops and tetraloop receptors, and incorporation of specific binding sites for other proteins (U1A or a recently developed RNA Fab) that can promote crystallization. We have also developed a rigorous strategy that will be used to ensure that each of these novel VA RNAI constructs retains wild-type PKR-inhibition activity, including global RNA folding analysis by UV melting, PKR autophosphorylation inhibition functional assays, and qualitative or quantitative measurement of PKR-RNA binding by gel filtration chromatography or isothermal titration calorimetry, respectively. Specific Aim 2 will employ high-throughput automated approaches to crystallization and X-ray diffraction screening to produce the necessary crystals. Initial experiments in this aim will be iterative in that first successes in crystallization or low resolution structure determination will feed back into the approaches of Aim 1 to refine the RNA construct design and ultimately produce crystals suitable for high-resolution X-ray crystal structure determination. The structure of the PKR-RNA complex will be determined by molecular replacement using the PKR kinase domain structure, other protein (U1A or Fab), and/or RNA fragments as the starting model(s). Alternatively, selenomethionine incorporation into protein components of the complex will be used to obtain experimental phases to determine the structure. These studies will thus provide a critical structural breakthrough that is a prerequisit for mechanistic studies of PKR and the future development of this important enzyme as a therapeutic target via structure aided design of small molecule inhibitors.

描述（由申请人提供）：PKR是针对病毒或细菌感染的细胞先天免疫反应的关键组成部分，是控制其他多种细胞过程的重要介体和积分因子，并具有对细胞周期，细胞生长，细胞凋亡以及对细胞应激的反应（如生长因子）的反应。异常PKR功能还与包括癌症，神经退行性疾病以及可能是代谢性疾病和肥胖症在内的人类疾病有关。我们对RNA或其他分子对PKR调节的理解及其作为药物靶标的潜力由于缺乏PKR-RNA复合物缺乏高分辨率结构信息而阻碍了它的潜力。该建议描述了一项研究计划，以获得双链RNA激活蛋白激酶（PKR）的晶体：非编码病毒RNA复合物，以生成这种重要的细胞激酶的第一个高分辨率分子快照及其调节RNA。我们应对这一主要挑战的方法分为两个综合目标。首先，由于RNA晶体学成功的最关键决定因素是RNA构建体本身，因此特定的目标1将利用我们对非编码腺病毒转录本VA RNAI的详细生化理解，以生成新型RNA构建体的多样化的“库”，以用PKR结晶。其中包括RNA结构结构域的系统变化，引入紧凑型和稳定的RNA二级结构，例如四边形和四边形受体，以及可以促进结晶的其他蛋白质（U1A或最近开发的RNA Fab）的特定结合位点（U1A或最近开发的RNA Fab）。我们还制定了一种严格的策略，该策略将用于确保这些新型的VA RNAi构造中的每一个都保留了野生型PKR抑制活性，包括通过紫外线熔化，PKR自磷酸化官能分析的全球RNA折叠分析，以及对PKR-RNA结合质量的定性或定性或定性或定性测量的质量量表或定性或定量测量。特定的目标2将采用高通量自动方法来结晶和X射线衍射筛选，以产生必要的晶体。该目标中的初始实验将是迭代的，因为结晶或低的首次成功分辨率结构的确定将反馈到AIM 1的方法中，以完善RNA构造设计，并最终产生适合高分辨率X射线晶体结构测定的晶体。 PKR-RNA复合物的结构将通过使用PKR激酶结构，其他蛋白质（U1A或FAB）和/或RNA片段作为起始模型来确定分子替代。或者，将硒甲氨基氨酸掺入复合物的蛋白质成分中，用于获得实验阶段以确定结构。因此，这些研究将提供一个关键的结构突破，这是PKR机械研究的先决条件以及该重要酶作为治疗靶靶标的未来发展，这是通过结构辅助小分子抑制剂的设计。