Mechanisms of RNA binding and remodeling proteins

RNA结合和重塑蛋白的机制

• 批准号: 7923334
• 负责人: CRAIG E. CAMERON
• 金额: $ 39.89万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7923334
• 关键词: Accounting; Address; Binding; Binding Proteins; Binding Sites; Biochemical; Biological; Biological Process; Boxing; Cells; Complex; Coupled; Crystallography; DNA-Directed RNA Polymerase; Data; Development; Dimerization; Disease; Double-Stranded RNA; Elements; Enzymes; Evaluation; Gene Expression Regulation; Genomics; Goals; Hepatitis C; Hepatitis C virus; Hydroxyl Radical; Infection; Intervention; Kinetics; Label; Lead; Liver Cirrhosis; Liver Fibrosis; Location; Malignant neoplasm of liver; Mass Spectrum Analysis; Medical; Metabolism; Methods; Modeling; Molecular; Molecular Motors; Molecular Structure; Mutate; Nucleic Acids; Pathway interactions; Play; Population; Protein Binding; Protein Footprinting; Proteins; RNA; RNA Binding; RNA Conformation; RNA Sequences; RNA Viruses; RNA replication; RNA-Binding Proteins; RNA-Protein Interaction; Replicon; Research Personnel; Research Proposals; Role; Site; Specificity; Structural Protein; Structure; Structure-Activity Relationship; System; Testing; Thermodynamics; Translations; Variant; Viral; Viral Proteins; Virus; Virus Diseases; Virus Replication; Work; abstracting; base; crosslink; helicase; in vivo; knowledge base; liver transplantation; melting; member; novel strategies; prevent; protein function; protein protein interaction; public health relevance; research study; therapy development; viral RNA

DESCRIPTION (provided by applicant): Mechanisms of RNA binding and remodeling proteins Abstract Manipulation of RNA requires the action of RNA binding proteins and ATP-dependent, molecular motor proteins that are believed to transport, remodel, and unwind secondary structures in RNA sequences. Many of these molecular motors are DEAD-box or closely related proteins. Positive strand RNA viruses such as the Hepatitis C virus (HCV) require the activity of these proteins for viral replication. Determination of the molecular mechanisms of these proteins is of fundamental importance to our understanding of RNA metabolism in general and will advance our understanding of how RNA virus' are replicated. The goal of this project is to determine the mechanism of RNA remodeling by an RNA binding protein and an RNA remodeling enzyme from the Hepatitis C virus. Non-structural protein 3 (NS3) is an RNA remodeling enzyme (or helicase), that is necessary for HCV replication. We propose a new model for RNA remodeling by this enzyme whereby multiple subunits of an oligomeric enzyme work together to melt out secondary structures in RNA. Our model predicts that NS3 participates in slow untwisting of the duplex in slow kinetic step, followed by rapid translocation in an ATP-dependent manner. We will test this model by using a combination of biophysical, biochemical, and biological experiments. We will identify the specific sites of protein-protein interactions by using protein footprinting coupled with mass spectrometry. Truncated forms of NS3 that do not form oligomeric structures will be examined to determine the specific roles that protein-protein interactions play in RNA remodeling. Our work has identified NS5A as an RNA binding protein that interacts with itself and with NS3. The structure of NS5A represents a new fold in nucleic acid recognition, and we are poised to uncover the structure/function relationship for RNA binding by this protein. The importance of protein-protein interactions will be determined by preparing variants of NS5A that are impeded in dimerization, followed by testing of those variants for RNA binding activity and for support of HCV replication in cells. The interplay between the NS3 and NS5A will be examined in detail using new biochemical and biological approaches. In aim 1, we will test our hypothesis for RNA remodeling by NS3. In aim 2, we will determine the role of dimerization of NS5A in RNA binding. In aim 3, the we will test the hypothesis that NS5A serves as a processivity factor for NS3 helicase activity. Determining the mechanisms of HCV RNA binding and remodeling proteins will reveal new molecular methods to disrupt the pathways responsible for HCV replication. Therefore, understanding the molecular mechanisms of proteins that bind and manipulate RNA is of biological and medical significance. PUBLIC HEALTH RELEVANCE: Mechanisms of RNA binding and remodeling proteins Narrative: Proteins that bind and manipulate RNA are of fundamental importance to many biological processes including translation, transcription, and gene regulation. RNA binding protiens also play key roles in viruses such as the Hepatitis C Virus, which infects almost 3 % of the world's population. Over 75% of HCV infections never resolve, resulting in persistent virus infection that can lead to liver fibrosis and, progressively, to severe and fatal diseases, including liver cirrhosis and liver cancer. Determining the mechanisms of HCV RNA binding and remodeling proteins will reveal new molecular methods to disrupt the pathways responsible for HCV replication. Therefore, understanding the molecular mechanisms of proteins that bind and manipulate RNA is of general biological and medical significance.

描述（由申请人提供）：RNA结合和重塑蛋白的机制抽象对RNA的操纵需要RNA结合蛋白和ATP依赖性的分子运动蛋白的作用，这些蛋白被认为可以在RNA序列中运输，重塑和二级结构。这些分子电动机中的许多是死箱或密切相关的蛋白质。阳性链RNA病毒（例如乙型肝炎病毒（HCV））需要这些蛋白质的活性以进行病毒复制。确定这些蛋白质的分子机制对于我们对RNA代谢的理解至关重要，并将促进我们对RNA病毒的复制方式的理解。该项目的目的是确定通过RNA结合蛋白和丙型肝炎病毒重塑酶的RNA重塑的机理。非结构蛋白3（NS3）是RNA重塑酶（或解旋酶），这对于HCV复制是必不可少的。我们提出了一种通过这种酶进行RNA重塑的新模型，从而使寡聚酶的多个亚基一起起作用，以融化RNA中的二级结构。我们的模型预测，NS3以缓慢的动力学步骤参与双链体的缓慢解开，然后以ATP依赖性方式快速易位。我们将通过结合生物物理，生化和生物学实验来测试该模型。我们将通过使用蛋白质足迹与质谱法结合蛋白质足迹来确定蛋白质蛋白质相互作用的特定位点。将检查不形成寡聚结构的NS3的截短形式，以确定蛋白质 - 蛋白质相互作用在RNA重塑中起的特定作用。我们的工作已将NS5A确定为与自身相互作用并与NS3相互作用的RNA结合蛋白。 NS5A的结构代表了核酸识别的新折叠，我们准备揭示该蛋白质结合RNA的结构/功能关系。蛋白质 - 蛋白质相互作用的重要性将通过制备二聚化障碍的NS5a的变体，然后测试这些变体的RNA结合活性，并支持细胞中HCV复制。 NS3和NS5A之间的相互作用将使用新的生化和生物学方法进行详细研究。在AIM 1中，我们将测试NS3对RNA重塑的假设。在AIM 2中，我们将确定NS5A在RNA结合中的二聚化作用。在AIM 3中，我们将测试NS5A作为NS3解旋酶活性的过程性因子的假设。确定HCV RNA结合和重塑蛋白的机制将揭示新的分子方法，以破坏负责HCV复制的途径。因此，了解结合和操纵RNA的蛋白质的分子机制具有生物学和医学意义。 公共卫生相关性：RNA结合和重塑蛋白叙事的机制：结合和操纵RNA的蛋白质对许多生物学过程至关重要，包括翻译，转录和基因调节。 RNA结合蛋白质还在诸如丙型肝炎病毒之类的病毒中起关键作用，该病毒感染了世界几乎3％的人口。超过75％的HCV感染永远无法解决，导致持续的病毒感染，可能导致肝纤维化，并逐渐导致严重和致命的疾病，包括肝硬化和肝癌。确定HCV RNA结合和重塑蛋白的机制将揭示新的分子方法，以破坏负责HCV复制的途径。因此，了解结合和操纵RNA的蛋白质的分子机制具有一般的生物学和医学意义。