Govindarajan Rajgopal, Leung George P H, Zhou Mingyan, Tse Chung-Ming, Wang Joanne, Unadkat Jashvant D
Department of Pharmaceutics, University of Washington, Box 357610, Seattle, WA 98195, USA.
Am J Physiol Gastrointest Liver Physiol. 2009 Apr;296(4):G910-22. doi: 10.1152/ajpgi.90672.2008. Epub 2009 Jan 22.
Human equilibrative nucleoside transporter-3 (hENT3) was recently reported as a pH-dependent, intracellular (lysosomal) transporter capable of transporting anti-human immunodeficiency virus (HIV) dideoxynucleosides (ddNs). Because most anti-HIV ddNs (e.g., zidovudine, AZT) exhibit clinical mitochondrial toxicity, we investigated whether hENT3 facilitates transport of anti-HIV ddNs into the mitochondria. Cellular fractionation and immunofluorescence microscopy studies in several human cell lines identified a substantial presence of hENT3 in the mitochondria, with additional presence at the cell surface of two placental cell lines (JAR, JEG3). Mitochondrial or cell surface hENT3 expression was confirmed in human hepatocytes and placental tissues, respectively. Unlike endogenous hENT3, yellow fluorescent protein (YFP)-tagged hENT3 was partially directed to the lysosomes. Xenopus oocytes expressing NH2-terminal-deleted hENT3 (expressed at the cell surface) showed pH-dependent interaction with several classes of nucleosides (anti-HIV ddNs, gemcitabine, fialuridine, ribavirin) that produce mitochondrial toxicity. Transport studies in hENT3 gene-silenced JAR cells showed significant reduction in mitochondrial transport of nucleosides and nucleoside drugs. Our data suggest that cellular localization of hENT3 is cell type dependent and the native transporter is substantially expressed in mitochondria and/or cell surface. hENT3-mediated mitochondrial transport may play an important role in mediating clinically observed mitochondrial toxicity of nucleoside drugs. In addition, our finding that hENT3 is a mitochondrial transporter is consistent with the recent finding that mutations in the hENT3 gene cause an autosomal recessive disorder in humans called the H syndrome.
人平衡核苷转运体3（hENT3）最近被报道为一种依赖pH的细胞内（溶酶体）转运体，能够转运抗人类免疫缺陷病毒（HIV）的双脱氧核苷（ddN）。由于大多数抗HIV的ddN（如齐多夫定，AZT）表现出临床线粒体毒性，我们研究了hENT3是否促进抗HIV的ddN转运到线粒体中。对几种人类细胞系进行的细胞分级分离和免疫荧光显微镜研究发现，线粒体中大量存在hENT3，在两种胎盘细胞系（JAR、JEG3）的细胞表面也有存在。分别在人肝细胞和胎盘组织中证实了线粒体或细胞表面hENT3的表达。与内源性hENT3不同，黄色荧光蛋白（YFP）标记的hENT3部分定位于溶酶体。表达NH2末端缺失的hENT3（在细胞表面表达）的非洲爪蟾卵母细胞显示出与几类产生线粒体毒性的核苷（抗HIV的ddN、吉西他滨、法匹拉韦、利巴韦林）存在pH依赖性相互作用。对hENT3基因沉默的JAR细胞进行的转运研究表明，核苷和核苷类药物的线粒体转运显著减少。我们的数据表明，hENT3的细胞定位取决于细胞类型，天然转运体在线粒体和/或细胞表面大量表达。hENT3介导的线粒体转运可能在介导核苷类药物临床上观察到的线粒体毒性中起重要作用。此外，我们发现hENT3是一种线粒体转运体，这与最近发现的hENT3基因突变导致人类常染色体隐性疾病H综合征的研究结果一致。
