Identification and functional analysis of new ribonuclease activated by death-receptor engagement

死亡受体结合激活的新型核糖核酸酶的鉴定和功能分析

基本信息

批准号：
13680727
负责人：
NADANO Daita
金额：
$ 1.09万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

项目摘要

This project started at the purification of ribonuclease activity, which is activated by death-receptor engagement, cleaves 28S ribosomal RNA specifically, inhibits protein synthesis, and finally promotes apoptosis. Using the lysate of apoptotic Jurkat cells obtained by the stimulation of a death receptor, Fas, and the normal, isotope (^<32>P)-labelled ribosome, conditions for cell-free analysis system were examined, and thus it was succeeded to reproduce in vitro the in vivo observation of 28S ribosomal RNA degradation and to analyze extensively the properties of the ribonuclese. Then, this work faced on a serious problem that the ribonuclease activity was inseparable from other intracellular non-specific ribonuclease activities by conventional biochemical, chromatographic methods. To solve this problem and analyze the relationship between apoptosis and ribosome further, new strategy has been adopted to identify proteins interacting the ribosome specifically in apoptotic cells by 1)isolation of ribosome fraction from apoptotic and normalcells,2)electrophoresis of proteins in both fractions to reveal a difference in patterns of protein bands,3)analysis of apoptosis-specific protein bands using mass spectrometry. This approach is comprehensive and is expected to enable us to identify not only the ribonuclease cleaving 28S ribosomal RNA but also other proteins which specifically interact with the ribosome in apoptotis.

该项目从核糖核酸酶活性的纯化开始，该酶通过死亡受体结合激活，特异性切割28S核糖体RNA，抑制蛋白质合成，最终促进细胞凋亡。使用通过刺激死亡受体Fas而获得的凋亡Jurkat细胞的裂解物和正常的同位素(^ 32 P)标记核糖体，检查了无细胞分析系统的条件，因此成功地体外重现 28S 核糖体 RNA 降解的体内观察结果，并广泛分析核糖核酸的特性。那么，本工作面临的一个严重问题是，通过常规的生化、色谱方法，核糖核酸酶活性与细胞内其他非特异性核糖核酸酶活性密不可分。为了解决这个问题并进一步分析细胞凋亡和核糖体之间的关系，采用了新的策略来鉴定凋亡细胞中与核糖体特异性相互作用的蛋白质，方法是：1）从凋亡细胞和正常细胞中分离核糖体组分，2）将两种组分中的蛋白质电泳到揭示蛋白质条带模式的差异，3）使用质谱分析凋亡特异性蛋白质条带。这种方法是全面的，预计使我们不仅能够识别切割 28S 核糖体 RNA 的核糖核酸酶，而且还能识别与细胞凋亡中的核糖体特异性相互作用的其他蛋白质。