The Molecular Mechanism Of Abnormal Neddylation Of Cullin3 Mutations In FHHt

Objective:Mutations in CUL3(skipping of exon 9,CUL3 ?9)could cause Familial hyperkalemic hypertension(FHHt).It was recently reported that CUL3 ?9 was more heavily neddylated and active than wild type CUL3,which would deplete its adptor protein KLHL3 strikingly and lead to WNK kinases accumulation.We will investigate whether abnormal neddylation of CUL3 ?9 is the result of its failure to bind to COP9 signalosome(CSN).Methods:HEK 293 cells were cultured in DMEM,high glucose,supplemented with 10% fetal bovine serum.FLAG-CUL3 or FLAGCUL3 ?9 plasmids were transfected into HEK 293 cells with Lipofectamine 2000,and then whole cell lysate was immunoprecipitated with Dynabeads Protein G after coating with FLAG M2 antibody.We could observe whether there was interaction between CSN and CUL3 or CUL3 ?9 by western blot.After transfection,we treated the HEK 293 cells with a metalloprotease inhibitor 1,10-phenanthroline or performed RNA interference technology to knock down CSN5,and then we could observe the change of neddylation level of CUL3 or CUL3 ?9 by western blot.Results:CUL3 ?9 showed markedly reduced binding to CSN5 according to the results of co-immunoprecipitation assay.Neddylation level of wild type CUL3 was increased after metalloprotease inhibitor 1,10-phenanthroline preincubation(p<0.05)or CSN5 RNAi(p<0.05).However,CUL3 ?9 neddylation level showed little change.Conclusion:Increased neddylation level of FHHt-causing Cullin3 mutations(CUL3 ?9)is likely the result of its failure to bind to CSN.