Proteomic Analysis of Eukaryotic Protein Synthesis

描述（由申请人提供）：这是一份长期项目的延续申请，该项目旨在全面鉴定和功能分析蛋白质相互作用和调节真核蛋白质合成的翻译后修饰。蛋白质合成是所有生物复制、生长和维持的基本过程。异常的蛋白质合成导致许多人类疾病，包括癌症和智力迟钝。因此，对正常和疾病改变的翻译过程进行系统分析需要对蛋白质合成有一个全面的了解。我们的策略将基于串联质谱的蛋白质组学与遗传和生化功能分析相结合，以鉴定和剖析未表征蛋白质的功能和控制翻译的翻译后修饰。最初，我们鉴定并验证了酿酒葡萄球菌中大量未鉴定的蛋白质和与核糖体、多体和翻译因子相关的翻译后修饰。在这个应用中，实验被提议来确定与翻译起始相关的这些蛋白质的一个子集的功能。起始是调控翻译的主要步骤。首先，我们将使用敏感的生化和遗传实验来检测酵母缺失突变体，以确定翻译起始的缺陷。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，以遗传鉴定相互作用的蛋白质。通过结合这些方法，我们期望为许多未表征的蛋白质开发功能模型。其次，我们将确定翻译起始因子的翻译后修饰。质谱法将用于特异性鉴定修饰的氨基酸。这些氨基酸将发生突变以确定其生物学意义。最后，我们将把我们的兴趣扩展到识别翻译起始因子，以了解不依赖帽的起始过程。必要的细胞周期基因鸟氨酸脱羧酶（ODC）和癌基因c- myc通过一种不依赖于帽的机制，利用内部核糖体进入位点（IRES）进行翻译。我们将使用新的蛋白质组学筛选来鉴定特异性结合这些mRNA位点的蛋白质。报告分析将用于测试候选蛋白对ODC和c-myc帽无关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.