Abstract
Equilibrative nucleoside transporters (ENTs) are important for the metabolic salvage of nucleosides and the cellular uptake of antineoplastic and antiviral nucleoside analogs. Human equilibrative nucleoside transporter 1 (hENT1) is inhibited by nanomolar concentrations of structurally diverse compounds, including dipyridamole, dilazep, nitrobenzylmercaptopurine ribonucleoside (NBMPR), draflazine, and soluflazine. Random mutagenesis and screening by functional complementation for inhibitor-resistant mutants in yeast revealed mutations at Phe-334 and Asn-338. Both residues are predicted to lie in transmembrane segment 8 (TM 8), which contains residues that are highly conserved in the ENT family. F334Y displayed increased V(max) values that were attributed to increased rates of catalytic turnover, and N338Q and N338C displayed altered membrane distributions that appeared to be because of protein folding defects. Mutations of Phe-334 or Asn-338 impaired interactions with dilazep and dipyridamole, whereas mutations of Asn-338 impaired interactions with draflazine and soluflazine. A helical wheel projection of TM 8 predicted that Phe-334 and Asn-338 lie in close proximity to other highly conserved and/or hydrophilic residues, suggesting that they form part of a structurally important region that influences interactions with inhibitors, protein folding, and rates of conformational change during the transport cycle.
Highlights
Nucleosides, which are hydrophilic molecules that do not readily diffuse across biological membranes, are the metabolic precursors of fundamental biological molecules such as DNA, RNA, and ATP
The aims of the work described in this study were to identify additional amino acid residues of Human equilibrative nucleoside transporter 1 (hENT1) involved in binding of dilazep, dipyridamole, and/or nitrobenzylmercaptopurine ribonucleoside (NBMPR) as well as residues involved in binding of draflazine and/or soluflazine
Yeast cells lack endogenous thymidine transport systems [41], production of recombinant hENT1 allows for salvage from the external medium of thymidine, which is phosphorylated to TMP by recombinant herpes simplex thymidine kinase in KTK yeast cells [35]
Summary
Strains and Media—KY114 (MAT␣, gal, ura, trp, lys, ade, hisd2000) was the parental yeast strain used to generate KTK, which produces recombinant herpes simplex thymidine kinase [30], and fui1::TRP1, which contains a disruption in the gene encoding the endogenous uridine permease (FUI1) [31]. Characterization of S. cerevisiae Membranes Containing Mutant hENT1 Proteins—Membranes were prepared from yeast cells harboring pYPhENT1 or mutants derived from this plasmid as described previously [35] in the presence of protease inhibitors (4-(2-aminoethyl)benzenesulfonyl fluoride, pepstatin A, E-64, and 1,10-phenanthroline) and protease inhibitor mixture for use with fungal and yeast extracts (Sigma). Their total protein content was measured using the bicinchoninic acid assay (Pierce). Apparent inhibition constants (Ki values) were calculated by the method of Cheng and Prusoff [39] from the observed IC50 values
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
