Identification of Structural and Molecular Features Involved in the Transport of 3'-Deoxy-Nucleoside Analogs by Human Equilibrative Nucleoside Transporter 3.
Identification of Structural and Molecular Features Involved in the Transport of 3'-Deoxy-Nucleoside Analogs by Human Equilibrative Nucleoside Transporter 3.
复制标题
人体平衡核苷转运蛋白 3 转运 3-脱氧核苷类似物所涉及的结构和分子特征的鉴定。
DOI：
10.1124/dmd.117.079400
复制
发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Govindarajan,Rajgopal
中科院分区：
文献类型：
--
作者：
Rahman,MdFazlur;Raj,Radhika;Govindarajan,Rajgopal
联合抗逆转录病毒药物治疗依赖于3′-脱氧核苷类似物，如3′-叠氮基-3 ′-脱氧胸苷（AZT）和2′3′-双脱氧肌苷（DDI）。尽管有效抑制人类免疫缺陷病毒复制，但这些药物产生一系列毒性，包括肌病、胰腺炎、神经病和乳酸性酸中毒，通常被认为是线粒体损伤的后遗症。虽然细胞表面定位的核苷转运蛋白，如人平衡型核苷转运蛋白2（hENT 2）和人浓缩型核苷转运蛋白1（hCNT 1），已知可增加载体介导的3′-脱氧核苷类似物进入细胞的摄取，但另一种广泛表达的细胞内核苷转运蛋白（即hENT 3）已涉及3′-脱氧核苷类似物的线粒体转运。通过定点突变、嵌合hENTs的产生以及突变体/嵌合RNA注射的非洲爪蟾卵母细胞中3 H渗透通量的测量，我们在此鉴定了决定3′-脱氧核苷类似物膜转位的hENT 3分子决定簇。我们的研究结果表明，hENT 1对3′-脱氧核苷类似物没有显著的转运活性，而hENT 3能够转运3′-脱氧核苷类似物，类似于hENT 2。hENT 3-hENT 1嵌合构建体的转运分析表明，hENT 3的N-末端的一半主要负责hENT 3 -3′-脱氧核苷类似物的相互作用。此外，致突变研究发现，hENT 3 N-末端一半的225 D和231 L部分有助于hENT 3转运AZT和DDI的能力。对3 '-脱氧核苷类似物的hENT 3转运中重要的转运蛋白片段和氨基酸残基的鉴定可能为克服与3'-脱氧核苷类似物治疗相关的不良毒性提供了一种可能的机制，并可能指导新型核苷类似物的合理开发。
Combination antiretroviral drug treatments depend on 3′-deoxy-nucleoside analogs such as 3′-azido-3′-deoxythymidine (AZT) and 2′3′-dideoxyinosine (DDI). Despite being effective in inhibiting human immunodeficiency virus replication, these drugs produce a range of toxicities, including myopathy, pancreatitis, neuropathy, and lactic acidosis, that are generally considered as sequelae to mitochondrial damage. Although cell surface–localized nucleoside transporters, such as human equilibrative nucleoside transporter 2 (hENT2) and human concentrative nucleoside transporter 1 (hCNT1), are known to increase the carrier-mediated uptake of 3′-deoxy-nucleoside analogs into cells, another ubiquitously expressed intracellular nucleoside transporter (namely, hENT3) has been implicated in the mitochondrial transport of 3′-deoxy-nucleoside analogs. Using site-directed mutagenesis, generation of chimeric hENTs, and 3H-permeant flux measurements in mutant/chimeric RNA–injected Xenopus oocytes, here we identified the molecular determinants of hENT3 that dictate membrane translocation of 3′-deoxy-nucleoside analogs. Our findings demonstrated that whereas hENT1 had no significant transport activity toward 3′-deoxy-nucleoside analogs, hENT3 was capable of transporting 3′-deoxy-nucleoside analogs similar to hENT2. Transport analyses of hENT3-hENT1 chimeric constructs demonstrated that the N-terminal half of hENT3 is primarily responsible for the hENT3–3′-deoxy-nucleoside analog interaction. In addition, mutagenic studies identified that 225D and 231L in the N-terminal half of hENT3 partially contribute to the ability of hENT3 to transport AZT and DDI. The identification of the transporter segment and amino acid residues that are important in hENT3 transport of 3′-deoxy-nucleoside analogs may present a possible mechanism for overcoming the adverse toxicities associated with 3′-deoxy-nucleoside analog treatment and may guide rational development of novel nucleoside analogs.