Université Paris Cité, French National Institute of Health and Medical Research (INSERM), Paris Cardiovascular Research Center (PARCC), F-75015 Paris, France.
Laboratory of Structural Biology of the Cell, Ecole Polytechnique, Institut Polytechnique de Paris, 91120 Palaiseau, France.
Int J Mol Sci. 2022 May 5;23(9):5151. doi: 10.3390/ijms23095151.
Cullin 3 (CUL3) is the scaffold of Cullin3 Ring E3-ligases (CRL3s), which use various BTB-adaptor proteins to ubiquitinate numerous substrates targeting their proteasomal degradation. 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.
Cullin 3（CUL3）是 Cullin3 环 E3 连接酶（CRL3s）的支架，它使用各种 BTB-接头蛋白来泛素化许多靶标蛋白使其进行蛋白酶体降解。导致家族性高钾血症和高血压（FHHt）的严重形式的突变，均导致外显子 9（氨基酸 403-459）缺失（CUL3-∆9）。令人惊讶的是，虽然 CUL3-∆9 高度发生类泛素化（一种通常激活 CRL 复合物的翻译后修饰），但其不能泛素化其底物。为了了解这种功能丧失的机制，我们通过质谱法对 CUL3 和 CUL3-∆9 相互作用组进行了比较无标记定量分析。观察到 CUL3-∆9 与 COP9 和 CAND1 的相互作用被破坏，这两种蛋白均参与 CRL3 复合物的动态组装。这些缺陷导致 CRL3 复合物的动态循环减少，使得 CRL3-∆9 复合物成为一种无活性的 BTB-接头陷阱，这一点通过 SILAC 实验得到证实。总的来说，数据表明，由于几个结构变化破坏了 CUL3-∆9 蛋白与关键调节伙伴的动态相互作用，导致高度类泛素化的 CUL3-∆9 蛋白失活。
