Department of Medicine, University of California, Irvine, California, USA.
Department of Environmental and Occupational Health, University of California, Irvine, California, USA.
J Nutr Biochem. 2023 Oct;120:109413. doi: 10.1016/j.jnutbio.2023.109413. Epub 2023 Jul 7.
The ubiquitin-proteasomal pathway regulates the functional expression of many membrane transporters in a variety of cellular systems. Nothing is currently known about the role of ubiquitin E3 ligase, neural precursor cell-expressed developmentally down-regulated gene 4 (Nedd4-1) and the proteasomal degradation pathway in regulating human vitamin C transporter-2 (hSVCT2) in neuronal cells. hSVCT2 mediates the uptake of ascorbic acid (AA) and is the predominantly expressed vitamin C transporter isoform in neuronal systems. Therefore, we addressed this knowledge gap in our study. Analysis of mRNA revealed markedly higher expression of Nedd4-1 in neuronal samples than that of Nedd4-2. Interestingly, Nedd4-1 expression in the hippocampus was higher in patients with Alzheimer's disease (AD) and age-dependently increased in the J20 mouse model of AD. The interaction of Nedd4-1 and hSVCT2 was confirmed by coimmunoprecipitation and colocalization. While the coexpression of Nedd4-1 with hSVCT2 displayed a significant decrease in AA uptake, siRNA-mediated knockdown of Nedd4-1 expression up-regulated the AA uptake. Further, we mutated a classical Nedd4 protein interacting motif ("PPXY") within the hSVCT2 polypeptide and observed markedly decreased AA uptake due to the intracellular localization of the mutated hSVCT2. Also, we determined the role of the proteasomal degradation pathway in hSVCT2 functional expression in SH-SY5Y cells and the results indicated that the proteasomal inhibitor (MG132) significantly up-regulated the AA uptake and hSVCT2 protein expression level. Taken together, our findings show that the regulation of hSVCT2 functional expression is at least partly mediated by the Nedd4-1 dependent ubiquitination and proteasomal pathways.
泛素蛋白酶体途径调节许多膜转运蛋白在各种细胞系统中的功能表达。目前尚不清楚泛素 E3 连接酶、神经前体细胞表达的发育下调基因 4（Nedd4-1）和蛋白酶体降解途径在调节神经元细胞中的人维生素 C 转运体 2（hSVCT2）中的作用。hSVCT2 介导抗坏血酸（AA）的摄取，是神经元系统中表达最广泛的维生素 C 转运体同工型。因此，我们在研究中解决了这一知识空白。mRNA 分析显示，神经元样本中 Nedd4-1 的表达明显高于 Nedd4-2。有趣的是，阿尔茨海默病（AD）患者海马中的 Nedd4-1 表达更高，并且在 AD 的 J20 小鼠模型中随年龄增长而增加。通过共免疫沉淀和共定位证实了 Nedd4-1 和 hSVCT2 的相互作用。虽然 Nedd4-1 与 hSVCT2 的共表达显著降低了 AA 的摄取，但 siRNA 介导的 Nedd4-1 表达下调则上调了 AA 的摄取。此外，我们在 hSVCT2 多肽内突变了一个经典的 Nedd4 蛋白相互作用基序（"PPXY"），并观察到由于突变的 hSVCT2 的细胞内定位，AA 的摄取明显减少。此外，我们还确定了蛋白酶体降解途径在 SH-SY5Y 细胞中 hSVCT2 功能表达中的作用，结果表明蛋白酶体抑制剂（MG132）显著上调了 AA 的摄取和 hSVCT2 蛋白表达水平。综上所述，我们的研究结果表明，hSVCT2 功能表达的调节至少部分是由 Nedd4-1 依赖性泛素化和蛋白酶体途径介导的。
