G418-mediated ribosomal read-through of a nonsense mutation causing autosomal recessive proximal renal tubular acidosis
G418-mediated ribosomal read-through of a nonsense mutation causing autosomal recessive proximal renal tubular acidosis
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DOI：
10.1152/ajprenal.00015.2008
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发表时间：
2008-09-01
影响因子：
4.2
通讯作者：
Kurtz, Ira
中科院分区：
文献类型：
--
作者：
Azimov, Rustam;Abuladze, Natalia;Kurtz, Ira
常染色体隐性近端肾小管酸中毒是由编码电致碳酸氢钠共转运体NBCe1-A的SLC4A4基因突变引起的。到目前为止，已被表征的突变会导致过早截断、错靶或降低共转运体的功能。尽管用碳酸氢盐治疗可纠正代谢性酸中毒，但肾外表现仍然存在，包括青光眼、白内障、角膜混浊和智力低下。目前，还没有已知的治疗方法可以专门针对突变的NBCe1-A蛋白。在本研究中，我们验证了NBCe1-A-Q29X突变可以通过氨基糖苷类抗生素在体外修复的假设，氨基糖苷类抗生素具有抑制过早停止密码子的能力。作为模型系统，我们将NBCe1-A-Q29X突变体克隆到缺乏氨基糖苷抗性基因的载体中，并将突变体共转运体转染到HEK293-H细胞中。转染NBCe1-A-Q29X突变体的细胞由于过早停止密码子而无法表达共转运蛋白。免疫印迹分析显示，用G418处理细胞可显著增加全长共转运蛋白的表达。此外，免疫细胞化学研究表明，G418处理诱导质膜上的共转运蛋白表达，而在没有G418的情况下，NBCe1-A-Q29X不表达。在未用G418处理的转染NBCe1-A-Q29X突变体的HEK293-H细胞中，未检测到nbce1 - a介导的通量。相比之下，在转染了NBCe1-A- q29x突变体的细胞中，G418处理诱导的Na+-和HCO3-依赖性转运与野生型NBCe1-A的功能没有区别。G418对模拟转染细胞无影响。综上所述，G418诱导HEK293-H细胞中NBCe1-A-Q29X突变的核糖体解读。这些发现首次证明，在引起近端肾小管酸中毒的NBCe1-A- q29x突变存在的情况下，可以产生全长功能性NBCe1-A蛋白。我们的研究结果首次证明了NBCe1-A的突变已经以靶向和特定的方式进行了治疗。
Autosomal recessive proximal renal tubular acidosis is caused by mutations in the SLC4A4 gene encoding the electrogenic sodium bicarbonate cotransporter NBCe1-A. The mutations that have been characterized thus far result in premature truncation, mistargeting, or decreased function of the cotransporter. Despite bicarbonate treatment to correct the metabolic acidosis, extrarenal manifestations persist, including glaucoma, cataracts, corneal opacification, and mental retardation. Currently, there are no known therapeutic approaches that can specifically target mutant NBCe1-A proteins. In the present study, we tested the hypothesis that the NBCe1-A-Q29X mutation can be rescued in vitro by treatment with aminoglycoside antibiotics, which are known for their ability to suppress premature stop codons. As a model system, we cloned the NBCe1-A-Q29X mutant into a vector lacking an aminoglycoside resistance gene and transfected the mutant cotransporter in HEK293-H cells. Cells transfected with the NBCe1-A-Q29X mutant failed to express the cotransporter because of the premature stop codon. Treatment of the cells with G418 significantly increased the expression of the full-length cotransporter, as assessed by immunoblot analysis. Furthermore, immunocytochemical studies demonstrated that G418 treatment induced cotransporter expression on the plasma membrane whereas in the absence of G418, NBCe1-A-Q29X was not expressed. In HEK293-H cells transfected with the NBCe1-A-Q29X mutant not treated with G418, NBCe1-A-mediated flux was not detectable. In contrast, in cells transfected with the NBCe1-A-Q29X mutant, G418 treatment induced Na+- and HCO3--dependent transport that did not differ from wild-type NBCe1-A function. G418 treatment in mock-transfected cells was without effect. In conclusion, G418 induces ribosomal read-through of the NBCe1-A-Q29X mutation in HEK293-H cells. These findings represent the first evidence that in the presence of the NBCe1-A-Q29X mutation that causes proximal renal tubular acidosis, full-length functional NBCe1-A protein can be produced. Our results provide the first demonstration of a mutation in NBCe1-A that has been treated in a targeted and specific manner.