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目的 探讨 p5 3的转录过程与其泛素调节的蛋白降解途径的相互关系。方法 采用瞬时转染报告基因测定法 ,测定细胞裂解液荧光素酶活性 (p5 3的转录活性 )或Western -blot、免疫共沉淀测定细胞内 p5 3蛋白水平。结果 删除 p5 3N末端 1- 19个氨基酸 (mdm2结合位点 ) ,可显著减少 p5 3的转录活性 ,提示p5 3的降解片段不能与其转录活性片段相分离。特异性蛋白酶体抑制剂存在时 ,细胞内 p5 3稳态蛋白水平升高 ,而 p5 3的转录活性受到显著的抑制。UBA酶 (ubiquitin -activatingenzyme)失活 ,导致p5 3转录功能降低。结论 p5 3的蛋白水解途径对其转录反应是必须的
Objective To investigate the relationship between p5 3 transcription and its ubiquitin-regulated protein degradation pathway. Methods The luciferase activity (transcript activity of p5 3) or Western-blot was determined by transient transfection reporter assay and the level of p5 3 protein was determined by co-immunoprecipitation. Results The deletion of 1 to 19 amino acids (the mdm2 binding site) at the 3N end of p5 significantly reduced the transcriptional activity of p5 3, suggesting that the degradation fragment of p5 3 could not be separated from its transcriptional active fragments. In the presence of specific proteasome inhibitors, the intracellular level of p5 <3> homeostasis protein increased whereas the transcription activity of p5 <3> was significantly inhibited. UBA enzyme (ubiquitin-activatingenzyme) inactivation, resulting in reduced transcription of p5 3 function. Conclusion The p5 3 proteolytic pathway is necessary for its transcriptional response