The search and analysis for essential signaling mediators responding to radiation by proteome techniques.

为了分离和鉴定对辐射反应的必要信号介质，我们搜索了辐射诱导的DNA损伤与细胞周期阻滞和细胞凋亡等细胞后果之间的信号通路的调节蛋白，并通过蛋白质组技术分析了它们的功能。近3年来取得的成果可以总结如下：1。我们研究了x射线照射胸腺细胞凋亡过程中染色体乘客蛋白（CPPs）亚细胞定位的改变，包括Aurora-B、survivin和INCENP。辐射暴露后，在洗涤不溶性部分和caspase-3裂解的INCENP中存在Aurora-B和survivin，提示CPPs复合物的异常形成导致细胞凋亡和染色体分离错误。通过蛋白质组学分析，我们发现RNA甲基转移酶NSUN2是Aurora-B的一个新的底物，并证实在有丝分裂过程中，Aurora-B通过磷酸化Ser139位点参与调节RNA甲基转移酶的活性。我们用二维凝胶电泳（2DE）分析了伽玛射线或紫外线照射的人白血病Jurkat细胞的细胞蛋白。在密集的蛋白斑点中，利用质谱技术鉴定了酸性核糖体蛋白P2 (RpP2)，发现RpP2的去磷酸化可能与辐射诱导Jurkat细胞凋亡有关。关于MDM2-MDMX复合物生物学特性的实验结果表明，MDM2和MDMX之间的相互作用代表了p53调控的一种新模式。这些结果表明，除了p53外，极光激酶和酸性核糖体蛋白在辐射诱导的信号转导中起重要作用。

To isolate and identify the essential signaling mediators responding to radiation, we have searched the proteins regulating signaling pathways between radiation-induced DNA damage and cellular consequences such as cell cycle arrest and apoptotic cell death, and analyzed their functions by proteome techniques. The results obtained for the past 3 years can be summarized as follows.1. We have examined alteration of the subcellular localization of chromosome passenger proteins (CPPs) including Aurora-B, survivin and INCENP during apoptosis in X-irradiated thymic cells. Aurora-B and survivin were present in detergent insoluble fraction and INCENP cleaved by caspase-3 after radiation exposure, suggesting that the abnormal formation of CPPs complex lead to the induction of apoptosis and chromosome segregation errors.2. We could identify the RNA methyltransferase NSUN2 as a novel substrate of Aurora-B by proteome analysis, and demonstrated that the Aurora-B participate to regulate the RNA methyltransferase activity via phosphorylation at Ser139 during mitosis.3. We have analyzed cellular proteins from human leukemic Jurkat cells irradiated with gamma-rays or UV using two dimensional gel electrophoresis (2DE). Among intensive protein spots, acidic ribosomal protein P2 (RpP2) was identified by peptide mass fingerprinting using a mass spectrometry and found that the dephosphorylation of RpP2 may be associated with induction of apoptosis in Jurkat cells by radiation.4. The results of experiments concerning biological properties of MDM2-MDMX complex suggest that the interaction between MDM2 and MDMX represents a novel mode of p53 regulation.These results suggest that Aurora kinases and acidic ribosomal proteins in addition to p53 play an important role in radiation-induced signal transduction.