Neddylation-mediated Nedd4-2 activation regulates ubiquitination modification of renal NBCe1
Neddylation-mediated Nedd4-2 activation regulates ubiquitination modification of renal NBCe1
复制标题
Neddylation 介导的 Nedd4-2 激活调节肾 NBCe1 的泛素化修饰。
DOI：
10.1016/j.yexcr.2020.111958
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发表时间：
2020-05-15
影响因子：
3.7
通讯作者：
Zhao，Yanyan
中科院分区：
文献类型：
--
作者：
Tu，Jianqiao;Zhang，Bijun;Zhao，Yanyan
钠偶联碳酸氢盐协同转运蛋白1（NBCe 1）在维持人体酸碱平衡方面发挥着重要作用。然而，关于NBCe 1的改性的研究很少。Nedd 4 -2是肾脏中最重要的泛素E3连接酶之一，负责介导许多重要离子通道蛋白的泛素化水平;因此，影响它们的表达和膜定位。在这项研究中，我们进行了实验的基础上，预测从生物信息学分析，NBCe 1可能是一个Nedd 4 -2的目标蛋白。免疫共沉淀和谷胱甘肽S-转移酶pull-down实验结果表明Nedd 4 -2与NBCe 1相互作用。Anin vitrobiquitination试验进一步证明Nedd 4 -2确实是NBCe 1泛素E3连接酶。Nedd 4 -2的过表达降低了NBCe 1的表达，而MG 132挽救了这些变化。Nedd 4 -2过表达也改变了NBCe 1的亚细胞分布。此外，肾脏特异性Nedd 4 -2基因敲除小鼠证实了NBCe 1的改变。此外，我们推测neddylation激活Nedd 4 -2。免疫共沉淀分析表明Nedd 4 -2与Nedd 8相互作用，体外neddylation实验进一步证明Nedd 4 -2发生了neddylation修饰。Nedd 8的过表达导致NBCe 1表达降低，而Nedd 4 -2抑制则挽救了这些变化。这些发现表明Nedd 4 -2作为NBCe 1的遍在蛋白E3连接酶，介导NBCe 1的降解并改变其亚细胞分布，并且neddylation修饰通过上调Nedd 4 -2活性来下调NBCe 1的表达。
The sodium-coupled bicarbonate cotransporter 1 (NBCe1) plays an essential role in the maintenance of acid-base homeostasis in the human body. However, little research has been done regarding the modification of NBCe1. Nedd4-2 is one of the most important ubiquitin E3 ligases in the kidney where it is responsible for mediating the ubiquitylation level of many important ion channel proteins; therefore, influencing their expression and membrane localization. In this study, we performed experiments based on a prediction from bioinformatics analysis that NBCe1 might be a Nedd4-2 target protein. The results of co-immunoprecipitation and glutathione S-transferase pull-down assays showed that Nedd4-2 interacted with NBCe1. Anin vitroubiquitination assay further demonstrated that Nedd4-2 is indeed the NBCe1 ubiquitin E3 ligase. The overexpression of Nedd4-2 decreased NBCe1 expression, while MG132 rescued the changes. Nedd4-2 overexpression also altered the subcellular distribution of NBCe1. Furthermore, the kidney specific Nedd4-2-knockout mice certified the alteration of NBCe1. In addition, we speculate that neddylation activates Nedd4-2. A co-immunoprecipitation analysis indicated that Nedd4-2 interacted with Nedd8.In vitroneddylation experiments further demonstrated that Nedd4-2 underwent neddylation modification. The overexpression of Nedd8 led to decreased NBCe1 expression, while Nedd4-2 inhibition rescued the changes. These findings demonstrate that Nedd4-2 acts as the ubiquitin E3 ligase of NBCe1, mediating the degradation and altering the subcellular distribution of NBCe1, and that the neddylation modification downregulated NBCe1 expression by upregulating Nedd4-2 activity.