Mitochondrial expression of the human equilibrative nucleoside transporter 1 (hENT1) results in enhanced mitochondrial toxicity of antiviral drugs
Mitochondrial expression of the human equilibrative nucleoside transporter 1 (hENT1) results in enhanced mitochondrial toxicity of antiviral drugs
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DOI：
10.1074/jbc.m307938200
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发表时间：
2004-02-06
影响因子：
4.8
通讯作者：
Unadkat, JD
中科院分区：
文献类型：
--
作者：
Lai, YR;Tse, CM;Unadkat, JD
许多抗病毒药物（如氟替尼定; FIAU）产生临床上显著的线粒体毒性，限制了它们的剂量或阻止它们在临床上的使用。由于大多数核苷类药物的亲水性太强，无法穿过高度不可渗透的线粒体膜，因此我们假设它们必须被转运到线粒体中才能产生毒性。为了验证这一假设，我们试图确定是否核苷转运蛋白，人平衡型核苷转运蛋白1（hENT 1）或人浓缩型核苷转运蛋白1（hCNT 1），当在Madin-Darby犬肾细胞中稳定表达为黄色荧光融合蛋白（YFP）时，定位于线粒体。通过使用细胞器选择性染料和共聚焦显微镜，我们已经发现，hENT 1-YFP是本地化的线粒体以及质膜，而hCNT 1-YFP被发现主要是在质膜上。hENT 1-YFP不定位于核膜、内体、溶酶体或高尔基复合体。蛋白质印迹法证实了hENT 1-YFP或内源性hENT 1的存在下，从hENT 1-YFP表达细胞和人肝脏中分离的线粒体，分别。与这些定位数据一致，[C-14]FIAU被有效地转运到表达hENT 1-YFP的细胞的线粒体中，但不是表达hCNT 1-YFP的细胞的线粒体中。FIAU对Madin-Darby犬肾细胞的线粒体毒性被hENT 1-YFP增强，即使当hENT 1在质膜上的活性被10 nM硝基苄基硫代肌苷选择性阻断时。此外，当FIAU（50 μ m）与从hENT 1表达细胞中分离的线粒体直接孵育时，FIAU（50 μ m）产生显著的线粒体毒性（类似于线粒体DNA合成减少70%）。总之，我们首次发现hENT 1在线粒体膜上表达，这种表达增强了FIAU等核苷类药物的线粒体毒性。hENTs的线粒体表达可以解释抗HIV核苷类药物如齐多夫定、司他夫定和去羟肌苷引起的临床显著的线粒体毒性。
Many antiviral drugs (e.g. fialuridine; FIAU) produce clinically significant mitochondrial toxicity that limits their dose or prevents their use in the clinic. Because the majority of nucleoside drugs is too hydrophilic to cross the highly impermeable mitochondrial membrane, we have hypothesized that they must be transported into the mitochondria to produce their toxicity. To test this hypothesis, we have sought to determine whether the nucleoside transporters, human equilibrative nucleoside transporter 1 (hENT1) or human concentrative nucleoside transporter 1 (hCNT1), when stably expressed in Madin-Darby canine kidney cells as yellow fluorescent fusion protein (YFP), are localized to the mitochondria. By using organelle-selective dyes and confocal microscopy, we have found that hENT1-YFP is localized to the mitochondria as well as the plasma membrane, whereas hCNT1-YFP was found predominantly on the plasma membrane. hENT1-YFP was not localized to the nuclear envelope, endosomes, lysosomes, or Golgi complex. Western blotting confirmed the presence of hENT1-YFP or endogenous hENT1 in mitochondria isolated from hENT1-YFP-expressing cells and human livers, respectively. In agreement with these localization data, [C-14]FIAU was efficiently transported into the mitochondria of cells expressing hENT1-YFP but not of cells expressing hCNT1-YFP. The mitochondrial toxicity of FIAU to Madin-Darby canine kidney cells was enhanced by hENT1-YFP, even when hENT1 activity on the plasma membrane was selectively blocked by 10 nM nitrobenzylthioinosine. Moreover, FIAU (50 mum) produced significant mitochondrial toxicity (similar to70% decrease in mitochondrial DNA synthesis) when it was directly incubated with mitochondria isolated from hENT1-expressing cells. In conclusion, we have identified for the first time that hENT1 is expressed on the mitochondrial membrane and that this expression enhances the mitochondrial toxicity of nucleoside drugs such as FIAU. Mitochondrial expression of hENTs may explain the clinically significant mitochondrial toxicity caused by the anti-HIV nucleoside drugs such as zidovudine, stavudine, and didanosine.