title: Functional characterization of human equilibrative nucleoside transporter 1.
abstract: 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 Kmof 215 ± 34 µmol/L and a Vmaxof 578 ± 23.4 nmol mg-1min-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.
authors.affiliation: Beijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, 100084, China.
authors.forename: Weiyun
authors.identifier: 0000-0003-2923-8592
authors.initials: W
authors.lastname: Huang
authors.affiliation: Beijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, 100084, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Zeng
authors.affiliation: Beijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, 100084, China.
authors.forename: Yigong
authors.initials: Y
authors.lastname: Shi
authors.affiliation: Beijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, 100084, China. liuminhao@biomed.tsinghua.edu.cn.
authors.forename: Minhao
authors.identifier: 0000-0002-9485-6955
authors.initials: M
authors.lastname: Liu
mesh_terms: D000227:Adenine Nucleotides / Q000737:chemistry* / Q000378:metabolism; D033721:Equilibrative Nucleoside Transporter 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D000072417:Protein Domains; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D000227:Adenine Nucleotides; D033721:Equilibrative Nucleoside Transporter 1; D011994:Recombinant Proteins; C438649:SLC29A1 protein, human
keywords: counter flow; functional characterization; hENT1; nucleoside; transport