Cullin 3 Exon 9 Deletion in Familial Hyperkalemic Hypertension Impairs Cullin3‐Ring‐E3 Ligase (CRL3) Dynamic Regulation and Cycling

Cullin 3 (CUL3) is the scaffold of Cullin3 Ring E3‐ligases (CRL3s), which use various BTBadaptor proteins to ubiquitinate numerous substrates targeting their proteasomal degradation. CUL3 mutations, responsible for a severe form of familial hyperkalemia and hypertension (FHHt), all result in a deletion of exon 9 (amino‐acids 403‐459) (CUL3‐∆9). Surprisingly, while CUL3‐∆9 is hyperneddylated, a post‐translational modification that typically activates CRL complexes, it is unable to ubiquitinate its substrates. In order to understand the mechanisms behind this loss‐of function, we performed comparative label‐free quantitative analyses of CUL3 and CUL3‐∆9 interactome by mass spectrometry. It was observed that CUL3‐∆9 interactions with COP9 and CAND1, both involved in CRL3 complexes’ dynamic assembly, were disrupted. These defects result in a reduction in the dynamic cycling of the CRL3 complexes, making the CRL3‐∆9 complex an inactive BTB‐adaptor trap, as demonstrated by SILAC experiments. Collectively, the data indicated that the hyperneddylated CUL3‐∆9 protein is inactive as a consequence of several structural changes disrupting its dynamic interactions with key regulatory partners.