家族性高血钾型高血压Cullin3致病突变体neddylation异常的分子机制研究

Molecular mechanism of abnormal neddylation of Cullin3 pathogenic mutant in familial hyperkalemic hypertension

目的·探讨Cullin3(CUL3)致病突变体Cullin3Δ9(缺失9号外显子,CUL3Δ9)是否由于与COP9信号体(CSN)结合减弱而导致其类泛素化修饰异常。方法·使用含10%胎牛血清的高糖DMEM培养基培养HEK293细胞株,采用脂质体转染技术,将CSN5 siRNA与FLAG-CUL3质粒或FLAG-CUL3Δ9质粒共转染HEK293细胞,Western blotting观察CUL3和CUL3Δ9类泛素化修饰程度的变化;转染FLAG-CUL3或FLAG-CUL3Δ9质粒后,以金属蛋白酶抑制剂1,10-菲咯啉进行预孵育,Western blotting观察CUL3和CUL3Δ9类泛素化修饰程度的变化;在转染FLAG-CUL3质粒和FLAG-CUL3Δ9质粒后,以FLAG抗体与Dynabeads Protein G磁珠行免疫共沉淀,Western blotting观察CUL3或CUL3Δ9与CSN5的结合状态。结果·转染CSN5 siRNA后,CUL3的类泛素化修饰程度增加,而CUL3Δ9的类泛素化修饰程度无明显变化;以1,10-菲罗啉预孵育后,CUL3的类泛素化修饰程度增加,而CUL3Δ9的类泛素化修饰程度无明显变化;免疫共沉淀结果显示CUL3与CSN5结合,而CUL3Δ9与CSN5结合减弱。结论·CUL3Δ9与CSN结合减弱导致类泛素化修饰异常。

Objective·To investigate whether abnormal neddylation of pathogenic mutant Cullin3 Δ9 （missing exon 9, i.e. CUL3 Δ9） is the result of reduced binding between CUL3 Δ9 and COP9 signalosome （CSN）. Methods·HEK 293 cells were cultured in high glucose DMEM containing 10% fetal bovine serum and were co-transfected with FLAG-CUL3 or FLAG-CUL3 Δ9 plasmids and CSN5 siRNA using liposome transfection technique. Changes of the neddylation level of CUL3 or CUL3 Δ9 were observed using Western blotting. After transfection of FLAG-CUL3 or FLAG-CUL3 Δ9 plasmids, HEK 293 cells were preincubated with metalloprotease inhibitor 1,10-phenanthroline. Change of neddylation level of CUL3 or CUL3 Δ9 were observed using Western blotting. After transfection of FLAG-CUL3 or FLAG-CUL3 Δ9 plasmids, co-immunoprecipitation was performed with FLAG antibody and Dynabeads Protein G. The binding between CSN5 and CUL3 or CUL3 Δ9 was observed using Western blotting. Results·Neddylation level of CUL3 was increased after transfection of CSN5 siRNA, while the neddylation level of CUL3 Δ9 showed little change. After preincubation with 1,10-phenanthroline, the neddylation level of CUL3 was increased, while the neddylation level of CUL3 Δ9 showed little change. Co-immunoprecipitation results showed that CUL3 bound with CSN5 and the binding between CUL3 Δ9 and CSN5 was reduced. Conclusion·Abnormal neddylation of pathogenic mutant Cullin3 Δ9 is the result of reduced binding between CUL3 Δ9 and CSN5.