Proteomic Analysis of Eukaryotic Protein Synthesis

描述(申请人提供)：这是一项长期项目的继续申请，旨在全面识别和功能分析调节真核蛋白质合成的蛋白质相互作用和翻译后修饰。蛋白质合成是所有生物复制、生长和维持所必需的基本过程。蛋白质合成异常会导致许多人类疾病，包括癌症和智力低下。因此，需要全面了解蛋白质的合成，以便对正常和疾病改变的翻译过程进行系统分析。我们的策略将基于串联质谱学的蛋白质组学与遗传和生化功能分析相结合，以识别和剖析未鉴定的蛋白质和控制翻译的翻译后修饰的功能。最初，我们在酿酒酵母中鉴定和验证了与核糖体、多聚体和翻译因子相关的大量未鉴定的蛋白质和翻译后修饰。在这一应用中，建议进行实验以确定与翻译启动相关的这些蛋白质的子集的功能。启动是规范翻译的主要步骤。首先，我们将使用敏感的生化和遗传学实验来检测酵母缺失突变体，以确定翻译启动过程中的缺陷。为了补充遗传和生化分析，我们将在全基因组范围内进行合成遗传分析，以从基因上识别相互作用的蛋白质。通过结合这些方法，我们希望为许多未表征的蛋白质开发功能模型。其次，我们将确定选定的翻译后修改对翻译启动因素的影响。质谱学将被用来专门鉴定修饰的氨基酸。这些氨基酸将发生突变，以确定它们的生物学意义。最后，我们将把我们对识别翻译启动因素的兴趣扩展到鲜为人知的非上限启动过程。重要的细胞周期基因鸟氨酸脱羧酶(ODC)和癌基因c-myc通过内部核糖体进入位点(IRES)通过帽不依赖的机制被翻译。我们将使用新的蛋白质组筛选来识别与这些mRNA位点特异结合的蛋白质。报告分析将被用来测试候选蛋白质对ODC和c-myc帽非依赖性翻译的积极和消极影响。

DESCRIPTION (provided by applicant): This is an application for continuation of a long-term project to comprehensively identify and functionally analyze protein interactions and posttranslational modifications regulating to eukaryotic protein synthesis. Protein synthesis is a fundamental process essential for replication, growth, and maintenance of all living organisms. Aberrant protein synthesis contributes to a number of human diseases including cancer and mental retardation. Hence a comprehensive understanding of protein synthesis is needed for systems analysis of both the normal and disease-altered translation process. Our strategy combines tandem mass spectrometry-based proteomics with genetic and biochemical functional assays to identify and dissect the function of uncharacterized proteins and posttranslational modifications controlling translation. Initially, we identified and validated a large number of uncharacterized proteins and posttranslational modifications associated with ribosomes, polysomes, and translation factors in S. cerevisiae. In this application, experiments are proposed to determine the function of a subset of these proteins linked to translation initiation. Initiation is the main step in which translation is regulated. First, we will assay yeast deletion mutants using sensitive biochemical and genetic experiments to identify defects in translation initiation. To complement the genetic and biochemical assays, we will perform synthetic genetic analysis on a genome- wide scale to genetically identify interacting proteins. By combining these approaches, we expect to develop functional models for many of uncharacterized proteins. Second, we will identify selected posttranslational modifications on translation initiation factors. Mass spectrometry will be used to specifically identify the modified amino acids. These amino acids will be mutated to establish their biological significance. Finally, we will extend our interest in identifying translation initiation factors to the poorly understood process of cap- independent initiation. The essential cell-cycle gene ornithine decarboxylase (ODC) and the oncogene c- myc are translated through a cap-independent mechanism using internal ribosomal entry sites (IRES). We will use novel proteomic screens to identify proteins that bind specifically to these mRNA sites. Reporter assays will be used be to test candidate proteins for positive and negative effects on ODC and c-myc cap- independent translation.