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

该项目从纯化核糖核酸酶活性开始，核糖核酸酶活性由死亡受体参与激活，特异性地切割28S核糖体RNA，抑制蛋白质合成，最终促进细胞凋亡。用死亡受体Fas刺激凋亡Jurkat细胞的裂解产物和正常同位素标记的核糖体，研究了建立无细胞分析系统的条件，从而成功地在体外复制了体内28S核糖体RNA降解的观察，并对核糖体的性质进行了广泛的分析。然后，本工作面临着一个严重的问题，即通过常规的生化、层析方法，核糖核酸酶活性与细胞内其他非特异性核糖核酸酶活性是分不开的。为了解决这一问题并进一步分析细胞凋亡与核糖体的关系，人们采用了一种新的策略来识别与核糖体相互作用的蛋白质，方法是：1)从凋亡细胞和正常细胞中分离核糖体部分，2)对这两个部分的蛋白质进行电泳以揭示不同的蛋白质条带，3)用质谱仪分析细胞凋亡特异的蛋白质条带。这种方法是全面的，有望使我们不仅能够识别核糖核酸酶裂解28S核糖体RNA，而且还能够识别在细胞凋亡中与核糖体特异相互作用的其他蛋白质。

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.