Functional characterization of human equilibrative nucleoside transporter 1.
Functional characterization of human equilibrative nucleoside transporter 1.
复制标题
人平衡核苷转运蛋白的功能表征1。
DOI：
10.1007/s13238-016-0350-x
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发表时间：
2017-04
期刊：
影响因子：
21.1
通讯作者：
Liu M
中科院分区：
文献类型：
--
作者：
Huang W;Zeng X;Shi Y;Liu M
平衡核苷转运蛋白（ENT）促进核苷和核苷衍生药物的跨膜转运，在核苷酸合成、癌症化疗和病毒感染治疗的挽救途径中发挥着重要作用。耳鼻喉科在分子水平上的功能表征在技术上仍然具有挑战性，因此很少。在这项研究中，我们报告了人平衡核苷转运蛋白 1 (hENT1) 的体外成功纯化和生化表征。 HEK293F 衍生的重组 hENT1 是同质的，并且在基于蛋白脂质体的逆流测定中具有功能活性。 hENT1 运输底物腺苷的 Km 为 215 ± 34 µmol/L，Vmax 为 578 ± 23.4 nmol mg−1 min−1。 hENT1 的腺苷摄取受到硝基苄基巯基嘌呤核糖核苷 (NBMPR)、核苷、脱氧核苷以及核苷衍生的抗癌和抗病毒药物的竞争性抑制。通过等温滴定量热法检测 hENT1 与腺苷、脱氧腺苷和腺嘌呤的结合与竞争性抑制测定的结果大体一致。这些结果验证了 hENT1 作为潜在药物靶点的真正靶点，并为未来的生物物理和结构研究提供了有用的基础。本文的在线版本 (doi:10.1007/s13238-016-0350-x) 包含补充材料，可供授权用户使用。
Equilibrative nucleoside transporters (ENTs), which facilitate cross-membrane transport of nucleosides and nucleoside-derived drugs, play an important role in the salvage pathways of nucleotide synthesis, cancer chemotherapy, and treatment for virus infections. Functional characterization of ENTs at the molecular level remains technically challenging and hence scant. In this study, we report successful purification and biochemical characterization of human equilibrative nucleoside transporter 1 (hENT1) in vitro. The HEK293F-derived, recombinant hENT1 is homogenous and functionally active in proteoliposome-based counter flow assays. hENT1 transports the substrate adenosine with a Km of 215 ± 34 µmol/L and a Vmax of 578 ± 23.4 nmol mg−1 min−1. Adenosine uptake by hENT1 is competitively inhibited by nitrobenzylmercaptopurine ribonucleoside (NBMPR), nucleosides, deoxynucleosides, and nucleoside-derived anti-cancer and anti-viral drugs. Binding of hENT1 to adenosine, deoxyadenosine, and adenine by isothermal titration calorimetry is in general agreement with results of the competitive inhibition assays. These results validate hENT1 as a bona fide target for potential drug target and serve as a useful basis for future biophysical and structural studies. The online version of this article (doi:10.1007/s13238-016-0350-x) contains supplementary material, which is available to authorized users.