Functional characterization of human equilibrative nucleoside transporter 1.
10.1007/s13238-016-0350-x
- Author:
Weiyun HUANG
1
;
Xin ZENG
1
;
Yigong SHI
1
;
Minhao LIU
2
Author Information
1. Beijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, 100084, China.
2. 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.
- Publication Type:Journal Article
- Keywords:
counter flow;
functional characterization;
hENT1;
nucleoside;
transport
- MeSH:
Adenine Nucleotides;
chemistry;
metabolism;
Equilibrative Nucleoside Transporter 1;
chemistry;
genetics;
metabolism;
HEK293 Cells;
Humans;
Protein Domains;
Recombinant Proteins;
chemistry;
genetics;
metabolism;
Structure-Activity Relationship
- From:
Protein & Cell
2017;8(4):284-295
- CountryChina
- Language:English
-
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 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.
