Huang Weiyun, Zeng Xin, Shi Yigong, Liu Minhao
Beijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, 100084, China.
Protein Cell. 2017 Apr;8(4):284-295. doi: 10.1007/s13238-016-0350-x. Epub 2016 Dec 19.
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 K of 215 ± 34 µmol/L and a V of 578 ± 23.4 nmol mg min. 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.
平衡核苷转运体（ENTs）促进核苷及核苷衍生药物的跨膜转运，在核苷酸合成的补救途径、癌症化疗及病毒感染治疗中发挥重要作用。在分子水平对ENTs进行功能表征在技术上仍具有挑战性，因此相关研究较少。在本研究中，我们报告了在体外成功纯化并对人平衡核苷转运体1（hENT1）进行生化表征。源自HEK293F的重组hENT1在基于蛋白脂质体的逆流测定中具有均一性且功能活跃。hENT1转运底物腺苷的K值为215±34μmol/L，V值为578±23.4nmol·mg-1·min-1。hENT1介导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测定hENT1与腺苷、脱氧腺苷和腺嘌呤的结合，结果与竞争性抑制试验结果总体一致。这些结果验证了hENT1作为潜在药物靶点的真实性，为未来的生物物理和结构研究提供了有用的基础。
