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Neddylation-mediated Nedd4-2 activation regulates ubiquitination modification of renal NBCe1.
Experimental Cell Research ( IF 3.7 ) Pub Date : 2020-03-12 , DOI: 10.1016/j.yexcr.2020.111958
Jianqiao Tu 1 , Bijun Zhang 1 , Guicun Fang 1 , Wenjing Chang 1 , Yanyan Zhao 1
Affiliation  

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. An in vitro ubiquitination 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 vitro neddylation 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.

中文翻译:

Neddylation介导的Nedd4-2激活调节肾脏NBCe1的泛素化修饰。

钠偶联的碳酸氢盐共转运蛋白1(NBCe1)在维持人体酸碱平衡方面起着至关重要的作用。但是,关于NBCe1的修饰研究很少。Nedd4-2是肾脏中最重要的泛素E3连接酶之一,它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,我们基于生物信息学分析的预测进行了实验,即NBCe1可能是Nedd4-2靶蛋白。免疫共沉淀和谷胱甘肽S-转移酶下拉实验的结果表明Nedd4-2与NBCe1相互作用。体外泛素化试验进一步证明Nedd4-2确实是NBCe1泛素E3连接酶。Nedd4-2的过表达降低了NBCe1表达,而MG132挽救了这些变化。Nedd4-2过表达也改变了NBCe1的亚细胞分布。此外,肾脏特异性Nedd4-2-敲除小鼠证明了NBCe1的改变。此外,我们推测,neddylation激活Nedd4-2。免疫共沉淀分析表明Nedd4-2与Nedd8相互作用。体外氰基化实验进一步证明Nedd4-2进行了氰基化修饰。Nedd8的过表达导致NBCe1表达降低,而Nedd4-2抑制则挽救了这种变化。这些发现表明Nedd4-2充当NBCe1的泛素E3连接酶,介导降解并改变NBCe1的亚细胞分布,
更新日期:2020-03-12
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