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

本项目从纯化核糖核酸酶活性开始，核糖核酸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。利用死亡受体Fas刺激获得的凋亡Jurkat细胞裂解液和正常同位素（^<32>P）标记的核糖体，研究了无细胞分析系统的条件，从而成功地在体外复制了28S核糖体RNA降解的体内观察并广泛分析了核糖体的性质。然后，本工作面临着常规生化、色谱方法无法与细胞内其他非特异性核糖核酸酶活性分离的严峻问题。为了解决这一问题并进一步分析细胞凋亡与核糖体之间的关系，我们采用了新的方法来鉴定凋亡细胞中与核糖体特异性相互作用的蛋白质：1)从凋亡细胞和正常细胞中分离核糖体片段，2)对两个片段中的蛋白质进行电泳以揭示蛋白质条带模式的差异，3)使用质谱法分析细胞凋亡特异性蛋白质条带。这种方法是全面的，有望使我们不仅能够鉴定核糖核酸酶切割28S核糖体RNA，而且还能鉴定凋亡中与核糖体特异性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.