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

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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.