NIH Undiagnosed Diseases Program, Common Fund, Office of the Director (H.E.C., L.A.W., T.C.M., D.R.A., W.A.G., P.S.), National Institutes of Health, Bethesda, MD.
Stem Cell Biochemistry Unit, National Institute of Dental and Craniofacial Research (J.C.C., A.W.), National Institutes of Health, Bethesda, MD.
Hypertension. 2022 Jan;79(1):60-75. doi: 10.1161/HYPERTENSIONAHA.121.17624. Epub 2021 Dec 2.
Familial hyperkalemic hypertension is caused by pathogenic variants in genes of the CUL3 (cullin-3)-KLHL3 (kelch-like-family-member-3)-WNK (with no-lysine [K] kinase) pathway, manifesting clinically as hyperkalemia, metabolic acidosis, and high systolic blood pressure. The ubiquitin E3 ligase CUL3-KLHL3 targets WNK kinases for degradation to limit activation of the thiazide-sensitive NCC (Na-Cl cotransporter). All known variants in lead to exon 9 skipping (CUL3Δ9) and typically result in severe familial hyperkalemic hypertension and growth disturbances in patients. Whether other variants in cause familial hyperkalemic hypertension is unknown. Here, we identify a novel de novo heterozygous variant (CUL3Δ474-477) in a pediatric familial hyperkalemic hypertension patient with multiple congenital anomalies and reveal molecular mechanisms by which CUL3Δ474-477 leads to dysregulation of the CUL3-KLHL3-WNK signaling axis. Using patient-derived urinary extracellular vesicles and dermal fibroblasts, in vitro assays, and cultured kidney cells, we demonstrate that CUL3Δ474-477 causes reduced total CUL3 levels due to increased autoubiquitination. The CUL3Δ474-477 that escapes autodegradation shows enhanced modification with NEDD8 (neural precursor cell expressed developmentally down-regulated protein 8) and increased formation of CUL3-KLHL3 complexes that are impaired in ubiquitinating WNK4. Proteomic analysis of CUL3 complexes revealed that, in addition to increased KLHL3 binding, the CUL3Δ474-477 variant also exhibits increased interactions with other BTB (Bric-a-brac, Tramtrack, and Broad complex) substrate adaptors, providing a rationale for the patient's diverse phenotypes. We conclude that the pathophysiological effects of CUL3Δ474-477 are caused by reduced CUL3 levels and formation of catalytically impaired CUL3 ligase complexes.
家族性高钾血症性高血压是由 CUL3（Cullin-3）-KLHL3（kelch 样家族成员 3）-WNK（无赖氨酸[K]激酶）通路基因的致病变异引起的，临床上表现为高钾血症、代谢性酸中毒和高收缩压。泛素 E3 连接酶 CUL3-KLHL3 将 WNK 激酶靶向降解，以限制噻嗪类敏感的 NCC（Na-Cl 共转运蛋白）的激活。已知的 中的所有变异都会导致外显子 9 跳跃（CUL3Δ9），通常导致患者严重的家族性高钾血症性高血压和生长障碍。 中的其他变异是否导致家族性高钾血症性高血压尚不清楚。在这里，我们在一名患有多种先天性异常的儿科家族性高钾血症性高血压患者中发现了一个新的从头杂合的 变异（CUL3Δ474-477），并揭示了 CUL3Δ474-477 导致 CUL3-KLHL3-WNK 信号轴失调的分子机制。使用患者来源的尿细胞外囊泡和真皮成纤维细胞、体外测定和培养的肾细胞，我们证明 CUL3Δ474-477 由于自身泛素化增加而导致总 CUL3 水平降低。逃避自降解的 CUL3Δ474-477 显示出与 NEDD8（神经前体细胞表达发育下调蛋白 8）的增强修饰以及 CUL3-KLHL3 复合物的形成增加，该复合物在泛素化 WNK4 方面受损。CUL3 复合物的蛋白质组学分析表明，除了 KLHL3 结合增加外，CUL3Δ474-477 变异体还表现出与其他 BTB（Bric-a-brac、Tramtrack 和 Broad complex）底物衔接蛋白的相互作用增加，为患者的多种表型提供了依据。我们得出结论，CUL3Δ474-477 的病理生理效应是由 CUL3 水平降低和催化失活的 CUL3 连接酶复合物形成引起的。
