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Analysis of the PKC-δ Signaling Pathway by Proteomics with Embryonic and Genetic Engeneering

胚胎和基因工程蛋白质组学分析 PKC-δ 信号通路

基本信息

批准号：
15370060
负责人：
NAKAYAMA Keiichi
金额：
$ 9.86万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

The accumulation of genome sequence data has facilitated the establishment of new approaches to systematic characterization of gene expression profiles at the mRNA level (transcriptome). Such strategies do not, however, necessarily provide direct information about the profile of protein expression (proteome), given that the abundance of a given mRNA does not necessarily correlate with that of the encoded protein. Furthermore, numerous characteristics of proteins, including their subcellular localization, interactions with other molecules, stability, and posttranslational modification, are amenable to study only at the protein level. Recent advances in methods for protein identification based on MS and searches of protein or DNA sequence databases have allowed high-throughput analysis of the proteome. Posttranslational modification, including phosphorylation, regulates the functions of proteins by affecting their interactions with other molecules, their enzymatic activity, or their subc … More ellular localization and is pivotal to the control of many cellular processes. Such modification is difficult to identify by standard proteomics approaches, however, because the modified proteins usually constitute a small proportion of all protein molecules. It is therefore necessary first to concentrate such modified proteins and to prevent contamination by highly abundant proteins. Highly sensitive MS analysis is then able to detect various types of posttranslational modification. Protein kinase C (PKC), which comprises 11 closely related isoforms, has been implicated in a wide variety of signaling mechanisms. Among PKC isotypes, PKC-δ is unique in that its overexpression results in inhibition of cell growth. We showed that mice that lack PKC-δ exhibit enlargement of peripheral lymphoid organs, expansion of the B lymphocyte population, as well as the presence of numerous germinal centers in lymphoid tissues in the absence of stimulation. We tried to develop a new approach designated "Proteomics with Embryonic and Genetic Engineering (PGEM)" to uncover the changes in PKC-δ-null mice. In this study, we established efficient and large-scale methods for phosphorylation and ubiquitylation of proteins. Furthermore, we also appled the SILAC method to quantitative analysis of the phosphoproteome. Less

基因组序列数据的积累促进了在mRNA水平（转录组）系统表征基因表达谱的新方法的建立。然而，这种策略不一定提供关于蛋白质表达谱（蛋白质组）的直接信息，因为给定mRNA的丰度不一定与编码蛋白质的丰度相关。此外，蛋白质的许多特性，包括它们的亚细胞定位、与其他分子的相互作用、稳定性和翻译后修饰，都只能在蛋白质水平上进行研究。基于MS和蛋白质或DNA序列数据库搜索的蛋白质鉴定方法的最新进展允许蛋白质组的高通量分析。翻译后修饰，包括磷酸化，通过影响蛋白质与其他分子的相互作用、酶活性或亚基活性来调节蛋白质的功能。 ...更多信息细胞定位，是控制许多细胞过程的关键。然而，这种修饰难以通过标准蛋白质组学方法鉴定，因为修饰的蛋白质通常构成所有蛋白质分子的一小部分。因此，必须首先浓缩这种修饰的蛋白质并防止被高丰度蛋白质污染。高灵敏度的MS分析能够检测各种类型的翻译后修饰。蛋白激酶C（PKC），包括11个密切相关的亚型，已被牵连在各种各样的信号转导机制。在PKC同种型中，PKC-δ是独特的，因为其过表达导致细胞生长抑制。我们发现，缺乏PKC-δ的小鼠表现出外周淋巴器官的扩大，B淋巴细胞群的扩增，以及在没有刺激的情况下淋巴组织中存在许多生发中心。我们尝试建立一种新的方法，称为“胚胎和基因工程蛋白质组学”（PGEM），以揭示PKC-δ-null小鼠的变化。在这项研究中，我们建立了高效和大规模的方法磷酸化和泛素化的蛋白质。此外，我们还将SILAC方法应用于磷酸化蛋白质组的定量分析。少