TRAF6对乳腺癌细胞中NHERF1蛋白稳定性影响的研究

Ubiquitin Ligase TRAF6 Regulates the Protein Stability of NHERF1 in Breast Cancer Cells

目的研究钠氢交换调控因子1（Na＋／H＋exchanger regulatory factor．1，NHERF1）的蛋白降解机制。方法采用蛋白酶体抑制剂MGl32处理乳腺癌细胞并检测NHERF1的蛋白和mRNA表达水平；过表达多种泛素连接酶并筛选特异性下调NHERF1蛋白表达水平的泛素连接酶；在乳腺癌细胞中梯度过表达TRAF6（TNF r-eceptorassociated factor6），检测TRAF6对NHERF1的蛋白和mRNA表达水平的影响；采用免疫荧光、免疫共沉淀和GST-pulldown实验验证NHERF1和TRAF6的相互作用。结果NHERF1蛋白的稳定性受泛素．蛋白酶体途径调控；TRAF6不影响NHERF1mRNA的表达水平，但可以通过其泛素连接酶活性下调NHERF1的蛋白表达水平；NHERF1与TRAF6两者之间存在相互作用。结论泛素连接酶TRAF6参与了NHERF1的泛素蛋白酶体途径降解。

Objective To explore the degradation mechanism of Na ＋/H＋ exchanger regulatory factor-1 （NHERF1） . Methods The proteasome inhibitor MG132 was used to treat breast cancer cells and the expression levels of NHERF1 protein and mRNA were detected. Over-expression of multiple ubiquitin ligases was performed toscreen of ubiquitin ligases that specifically downregulate NHERF1 protein expression level. TRAF6 was transfected into breast cancer cells to detectethe effects on the expression levels of NHERF1 protein and mRNA. The interaction between NHERF1 and TRAF6 was verified by immunofluorescence, co-immunoprecipitation and GST-pull down. Results Stability of NHERF1 was regulated byubiquitin-proteasome system （ UPS）, TRAF6 can down-regulate NHERF1 protein expression without affecting its mRNA expression level, NHERF1 interacts with TRAF6. Conclusion The ubiquitin ligase TRAF6 is involved in the degradation of NHERF1 through ubicluitin proteasome system.