Role of the SrmB Helicase in Ribosome Assembly investigated by Mass Spectrometry

通过质谱法研究 SrmB 解旋酶在核糖体组装中的作用

• 批准号: 8398150
• 负责人: CARLA F CERVANTES
• 金额: $ 5.22万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8398150
• 关键词: Affinity Chromatography; Amylose; Binding; Biogenesis; Boxing; Cells; Complex; Data; Diamond-Blackfan anemia; Disease; Dyskeratosis Congenita; Equipment and supply inventories; Escherichia coli; Family; Goals; Growth; Hereditary Disease; Incubated; Individual; Investigation; Knowledge; Label; Lead; Malignant Neoplasms; Mass Spectrum Analysis; Metabolic; Metabolism; Methodology; Methods; Molecular; Operon; Organism; Other Genetics; Pharmacotherapy; Physiologic pulse; Plant Resins; Plasmids; Process; Protein Binding; Protein Family; Proteins; Proteomics; Protocols documentation; RNA; RNA Helicase; Reporting; Ribosomal Proteins; Ribosomes; Role; Sampling; Shotguns; Sucrose; System; Ultracentrifugation; Virus; cancer cell; cancer genetics; cell growth; cofactor; design; effective therapy; helicase; human disease; in vivo; mutant; overexpression; particle; protein expression; research study; stable isotope

DESCRIPTION (provided by applicant): SrmB is an RNA helicase from the DEAD-box family of proteins that has a known function assisting in the early steps of 50S ribosome subunit assembly. DEAD-box proteins are known to be involved in nearly every step of RNA metabolism; however, their specific mechanisms of action are largely unknown. The goal of the proposed project is to investigate the mechanism by which SrmB assists in 50S ribosome subunit assembly in vivo via quantitative mass spectrometry (QMS). First, we will use three QMS experiments (protein inventory, pulse labeling, and discovery proteomics) to characterize ribosome assembly intermediates of the ?SrmB E.coli strain, fractionally separated by sucrose gradient ultracentrifugation. These experiments will report on, respectively, the ribosomal protein composition of the intermediates compared to an intact ribosome internal standard, whether the intermediates are true on path-intermediates or degradation products, and their ribosome biogenesis cofactor composition. We will perturb the system by introducing plasmids expressing WT and mutant SrmB in the ?SrmB strain background and analyze the sucrose gradient fractions of these via the aforementioned QMS experiments. Second, we will introduce WT and mutant MBP-SrmB expression plasmids into ?SrmB cells and perform MBP tag pull downs with amylose resin of both crude lysate and sucrose gradient fractions in order to purify MBP-SrmB-associated ribosomal particles. Elution fractions will be analyzed by QMS, as described before, which will give a characterization of SrmB-associated ribosomal intermediates. Third, we will investigate the relationship between SrmB and other ribosomal proteins by perturbing ribosomal protein expression by overexpression in ?SrmB E. coli cells of ribosomal proteins S4 or S8, which are known autoregulators of polycistrionic operons for ribosomal proteins. Sucrose gradient fractions of these growths will be analyzed by QMS, and this analysis will allow us to determine functional connections between binding of specific ribosomal proteins and SrmB. All together, the experiments proposed in the three aims of this proposal will give a detailed picture of 50S subunit assembly from the standpoint of SrmB: that is, what ribosomal proteins and cofactors are required for SrmB to bind the 50S precursor particle, which require SrmB in order to engage with the 50S precursor particle, and which ribosomal proteins and cofactors bind and act upon the 50S precursor particle independently of SrmB. Both, misregulation of, the fundamental process of ribosome biogenesis and overexpression of DEAD-box proteins are observed in various disease states, including cancer. The studies proposed here will lead to a deeper and more detailed understanding of the molecular mechanism whereby RNA helicases operate on their targets as well as the complex process of ribosome biogenesis, which is paramount for the design of effective therapies for diseases in which they are implicated. PUBLIC HEALTH RELEVANCE: Ribosome assembly is one of the major metabolic activities in cells and is required for cell growth in all organisms; not surprisingly, substantial evidence points to the misassembly of ribosomes as a major cause of human disease, including cancer and other genetic diseases such as Diamond-blackfan anemia and the severe genetic disorder dyskeratosis congenita. DEAD-box proteins are overexpressed in various types of cancer cells and RNA helicases from related families are essential for the propagation of many viruses that cause human diseases. The project proposed here will help further knowledge of the mechanism of SrmB and DEAD-box helicases in general, as well as the process of ribosome assembly, which can be used to design effective drugs and therapies for diseases in which they are implicated.

描述（由申请人提供）：SrmB 是一种来自 DEAD-box 蛋白质家族的 RNA 解旋酶，其已知功能有助于 50S 核糖体亚基组装的早期步骤。已知 DEAD-box 蛋白参与 RNA 代谢的几乎每一步；然而，它们的具体作用机制在很大程度上尚不清楚。该项目的目标是通过定量质谱 (QMS) 研究 SrmB 在体内协助 50S 核糖体亚基组装的机制。首先，我们将使用三个 QMS 实验（蛋白质库存、脉冲标记和发现蛋白质组学）来表征 ?SrmB 大肠杆菌菌株的核糖体组装中间体，通过蔗糖梯度超速离心进行分级分离。这些实验将分别报告与完整核糖体内标相比的中间体的核糖体蛋白质组成、中间体在路径中间体或降解产物上是否真实，以及它们的核糖体生物合成辅因子组成。我们将通过在 ?SrmB 菌株背景中引入表达 WT 和突变体 SrmB 的质粒来干扰系统，并通过上述 QMS 实验分析这些的蔗糖梯度分数。其次，我们将WT和突变MBP-SrmB表达质粒引入?SrmB细胞，并用粗裂解物和蔗糖梯度组分的直链淀粉树脂进行MBP标签下拉，以纯化MBP-SrmB相关核糖体颗粒。如前所述，洗脱组分将通过 QMS 进行分析，这将给出 SrmB 相关核糖体中间体的表征。第三，我们将通过在 ?SrmB 大肠杆菌细胞中过表达核糖体蛋白 S4 或 S8（已知的核糖体蛋白多顺反子操纵子的自动调节剂）来干扰核糖体蛋白表达，从而研究 SrmB 和其他核糖体蛋白之间的关系。这些生长物的蔗糖梯度部分将通过 QMS 进行分析，这种分析将使我们能够确定特定核糖体蛋白与 SrmB 结合之间的功能联系。总之，本提案的三个目标中提出的实验将从SrmB的角度详细描述50S亚基组装：即SrmB结合50S前体颗粒需要哪些核糖体蛋白和辅因子，哪些核糖体蛋白和辅因子需要SrmB才能与50S前体颗粒结合，以及哪些核糖体蛋白和辅因子结合并作用于50S前体 粒子独立于SrmB。在包括癌症在内的各种疾病状态中都观察到核糖体生物合成基本过程的错误调节和 DEAD-box 蛋白的过度表达。这里提出的研究将导致人们更深入、更详细地了解 RNA 解旋酶作用于其靶标的分子机制以及核糖体生物发生的复杂过程，这对于设计针对其所涉及的疾病的有效疗法至关重要。 公共卫生相关性：核糖体组装是细胞中的主要代谢活动之一，是所有生物体细胞生长所必需的；毫不奇怪，大量证据表明核糖体的错误组装是人类疾病的主要原因，包括癌症和其他遗传疾病，如钻石黑扇贫血症和严重的遗传性疾病先天性角化不良。 DEAD-box 蛋白在各种类型的癌细胞中过度表达，来自相关家族的 RNA 解旋酶对于许多引起人类疾病的病毒的传播至关重要。这里提出的项目将有助于进一步了解 SrmB 和 DEAD-box 解旋酶的一般机制，以及核糖体组装过程，这可用于设计针对它们所涉及的疾病的有效药物和疗法。