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已经准备好建立了在mRNA水平（转录组）上系统表征的新方法。但是，考虑到给定mRNA的抽象不一定与编码蛋白质的抽象相关，因此这种策略并不需要提供有关蛋白质表达（蛋白质组）的直接信息。此外，蛋白质的许多特征，包括其亚细胞定位，与其他分子的相互作用，稳定性和翻译后修饰，仅适合于蛋白质水平研究。基于MS的蛋白质鉴定方法的最新进展以及对蛋白质或DNA序列数据库的搜索允许对蛋白质进行高通量分析。翻译后修饰，包括磷酸化，通过影响其与其他分子的相互作用，酶活性或其SUBC的相互作用来调节蛋白质的功能……更椭圆形的定位，并且对控制许多细胞过程的控制至关重要。但是，由于改性蛋白通常构成所有蛋白质分子的一小部分，因此很难通过标准蛋白方法识别这种修饰。因此，首先有必要将这种改良的蛋白质浓缩并防止高度丰富的蛋白质污染。然后，高度敏感的MS分析能够检测到各种类型的翻译后修饰。蛋白激酶C（PKC）包括11种密切相关的同工型，已在多种信号传导机制中实施。在PKC同种型中，PKC-δ是独一无二的，因为其过表达会导致细胞生长的抑制。我们表明，缺乏PKC-δ的小鼠展示了周围淋巴器官的扩张，B淋巴细胞群的扩张以及在没有刺激的情况下在淋巴组织中存在许多生发中心。我们试图开发一种指定“具有胚胎和基因工程（PGEM）蛋白质组学的新方法”，以揭示PKC-δ-null小鼠的变化。在这项研究中，我们建立了蛋白质磷酸化和泛素化的有效和大规模方法。此外，我们还将SILAC方法添加到磷蛋白组的定量分析。较少的