Abstract:

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.

Key words: neddylation, Cullin3, COP9 signalosome, familial hyperkalemic hypertension