scholarly journals A Toxoplasma gondii Oxopurine Transporter Binds Nucleobases and Nucleosides Using Different Binding Modes

2022 ◽  
Vol 23 (2) ◽  
pp. 710
Author(s):  
Gustavo D. Campagnaro ◽  
Hamza A. A. Elati ◽  
Sofia Balaska ◽  
Maria Esther Martin Abril ◽  
Manal J. Natto ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
De Novo ◽  
Nucleoside Transporter ◽  
Binding Modes ◽  
Purine Analogues ◽  
Equilibrative Nucleoside Transporter ◽  
High Affinity ◽  
Purine Ring ◽  
Hydroxy Groups ◽  
Similar Affinity

Toxoplasma gondii is unable to synthesize purines de novo, instead salvages them from its environment, inside the host cell, for which they need high affinity carriers. Here, we report the expression of a T. gondii Equilibrative Nucleoside Transporter, Tg244440, in a Trypanosoma brucei strain from which nucleobase transporters have been deleted. Tg244440 transported hypoxanthine and guanine with similar affinity (Km ~1 µM), while inosine and guanosine displayed Ki values of 4.05 and 3.30 µM, respectively. Low affinity was observed for adenosine, adenine, and pyrimidines, classifying Tg244440 as a high affinity oxopurine transporter. Purine analogues were used to probe the substrate-transporter binding interactions, culminating in quantitative models showing different binding modes for oxopurine bases, oxopurine nucleosides, and adenosine. Hypoxanthine and guanine interacted through protonated N1 and N9, and through unprotonated N3 and N7 of the purine ring, whereas inosine and guanosine mostly employed the ribose hydroxy groups for binding, in addition to N1H of the nucleobase. Conversely, the ribose moiety of adenosine barely made any contribution to binding. Tg244440 is the first gene identified to encode a high affinity oxopurine transporter in T. gondii and, to the best of our knowledge, the first purine transporter to employ different binding modes for nucleosides and nucleobases.

Identification and Effects of Novel Promoter Region Haplotypes in the Human Equilibrative Nucleoside Transporter, hENT1.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2083-2083
Author(s):  
Scott N. Myers ◽  
Rakesh K. Goyal ◽  
Jennifer D. Roy ◽  
Robert E. Ferrell
Keyword(s):  
De Novo ◽  
Remission Rate ◽  
Leukemic Cells ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Nucleoside Transporter ◽  
Nucleotide Polymorphisms ◽  
Flanking Sequence ◽  
Equilibrative Nucleoside Transporter

Abstract Front-line induction chemotherapy regimens containing cytosine arabinoside (Ara-C) and anthracyclines result in 80% complete remission rate in childhood acute myeloid leukemia (AML) but their cure rate is about 35 – 50%, one of the lowest of all childhood cancers. Understanding the factors that contribute to emergence of chemoresistant leukemic cells is crucial to improving treatment outcome in children with AML. We are interested in studying the role of variation in Ara-C transport and biotransformation pathway genes in the efficacy and toxicity of treatment of childhood AML. To permeate the cell membrane, Ara-C is mainly dependent on human equilibrative nucleoside transporter 1 (hENT1; SLC29A1; gene localized to 6p21.1). Several studies have suggested an important role for altered levels of hENT1 in the chemosensitivity of AML blasts to Ara-C (Galmarini et al. Leukemia2001; 15(6):87; Gati et al. Leuk Lymphoma1998; 32(1–2):45). Osato and colleagues identified two single nucleotide polymorphisms (SNPs) in the hENT1 coding sequence that led to missense changes, but their in vitro analysis did not detect differences in the activity of variant alleles in a yeast transfection system (Osato et al. Pharmacogenetics2003;13(5):297). To identify variation in hENT1 that might influence its expression, we sequenced 1.6Kb of the proximal 5′-flanking sequence of the gene in 42 unrelated individuals and identified three SNPs at positions C-1345G, G-1050A, and G-706C. TRANSFAC analysis (www.genomatix.de) predicted that two of these (C-1345G & G-706C) would alter consensus transcription factor binding site sequences. We cloned four naturally occurring haplotypes (CGG, CAG, CGC, and GAG) using the TOPO-TA cloning kit, then transfected Cos-1 cells using the Lipofectamine 2000 protocol. Gene expression was assayed using the Promega Dual-Luciferase Reporter Assay System and read on a Molecular Devices HT Analyzer. Luciferase activity was measured at 24 and 48 hours after transfection for six replicates of every condition during three separate transfections. To correct for differences in transfection efficiencies, experimental (Photinus pyralis) luciferase activities were normalized by co-transfection with control (Renilla reniformis) luciferase plasmid. Compared to the wild type CGG haplotype, variant haplotypes CAG, CGC, and GAG drive luciferase expression at approximately 2x (p <0.0001), 1.4x (p <0.001) and 1.2x (p =0.08), respectively. This leads to the hypothesis that individuals carrying CAG or CGC haplotypes (17% of the population) exhibit higher levels of hENT1 expression and are more sensitive to Ara-C exposure. Experiments are underway to quantify gene transcripts in people of known hENT1 haplotypes. We also plan to genotype a large cohort of children with de novo AML for these three SNPs in hENT1 and correlate clinical outcomes in individuals carrying the low- versus the high-expressing haplotypes.

scholarly journals Purine Nucleobase Transport in Amastigotes of Leishmania mexicana: Involvement in Allopurinol Uptake

2005 ◽  
Vol 49 (9) ◽  
pp. 3682-3689 ◽  
Author(s):  
Mohammed I. Al-Salabi ◽  
Harry P. de Koning
Keyword(s):  
Leishmania Major ◽  
De Novo ◽  
Nucleoside Transporters ◽  
Human Pathogens ◽  
Leishmania Mexicana ◽  
Purine Analogues ◽  
Purine Salvage ◽  
High Affinity ◽  
Identification And Characterization

ABSTRACT Nucleobase and nucleoside transporters play central roles in the biochemistry of parasitic protozoa, as they lack the ability to synthesize purines de novo and are absolutely reliant upon purine salvage from their hosts. Furthermore, such transporters are potentially critical to the pharmacology of these important human pathogens, because they mediate the uptake of purine analogues, as well as some nonpurine drugs, that can be selectively cytotoxic to the parasites. We here report the first identification and characterization of a purine nucleobase transporter in Leishmania amastigotes. Uptake of [3H]hypoxanthine by Leishmania mexicana amastigotes was mediated by a single high-affinity transporter, LmexNBT1, with a Km of 1.6 ± 0.4 μM and high affinity for adenine, guanine, and xanthine but low affinity for nucleosides and pyrimidine nucleobases. Allopurinol, an antileishmanial hypoxanthine analogue, was apparently taken up by the same transporter. Using [3H]allopurinol, a Km value of 33.6 ± 6.0 μM was obtained. All evidence was compatible with a model of a single purine nucleobase transporter being expressed in amastigotes. Using various purine nucleobase analogues, a model for the interactions between hypoxanthine and the transporter's permeant binding site was constructed. The binding interactions were compared with those of the LmajNBT1 transporter in Leishmania major promastigotes and found to be very similar.

scholarly journals A comprehensive model of purine uptake by the malaria parasite Plasmodium falciparum: identification of four purine transport activities in intraerythrocytic parasites

2008 ◽  
Vol 411 (2) ◽  
pp. 287-295 ◽  
Author(s):  
Neils B. Quashie ◽  
Dominique Dorin-Semblat ◽  
Patrick G. Bray ◽  
Giancarlo A. Biagini ◽  
Christian Doerig ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
De Novo ◽  
High Capacity ◽  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Transport Activity ◽  
High Affinity ◽  
Parasite Plasmodium Falciparum ◽  
Adenosine Transport ◽  
Efficient Uptake

Plasmodium falciparum is incapable of de novo purine biosynthesis, and is absolutely dependent on transporters to salvage purines from the environment. Only one low-affinity adenosine transporter has been characterized to date. In the present study we report a comprehensive study of purine nucleobase and nucleoside transport by intraerythrocytic P. falciparum parasites. Isolated trophozoites expressed (i) a high-affinity hypoxanthine transporter with a secondary capacity for purine nucleosides, (ii) a separate high-affinity transporter for adenine, (iii) a low-affinity adenosine transporter, and (iv) a low-affinity/high-capacity adenine carrier. Hypoxanthine was taken up with 12-fold higher efficiency than adenosine. Using a parasite clone with a disrupted PfNT1 (P. falciparum nucleoside transporter 1) gene we found that the high-affinity hypoxanthine/nucleoside transport activity was completely abolished, whereas the low-affinity adenosine transport activity was unchanged. Adenine transport was increased, presumably to partly compensate for the loss of the high-affinity hypoxanthine transporter. We thus propose a model for purine salvage in P. falciparum, based on the highly efficient uptake of hypoxanthine by PfNT1 and a high capacity for purine nucleoside uptake by a lower affinity carrier.

scholarly journals Characterization of three novel members of the Arabidopsis thaliana equilibrative nucleoside transporter (ENT) family

2004 ◽  
Vol 383 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Alexandra WORMIT ◽  
Michaela TRAUB ◽  
Martin FLÖRCHINGER ◽  
H. Ekkehard NEUHAUS ◽  
Torsten MÖHLMANN
Keyword(s):  
Fluorescent Protein ◽  
De Novo ◽  
Biochemical Properties ◽  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Protein Fusion ◽  
Equilibrative Nucleoside Transporter ◽  
Transporter Family ◽  
Ph Dependency ◽  
Green Fluorescent

Research on metabolism of nucleotides and their derivatives has gained increasing interest in the recent past. This includes de novo synthesis, analysis of salvage pathways, breakdown and transport of nucleotides, nucleosides and nucleobases. To perform a further step towards the analysis of nucleoside transport in Arabidopsis, we incubated leaf discs with various radioactively labelled nucleosides. Leaf cells imported labelled nucleosides and incorporated these compounds into RNA, but not into DNA. Furthermore, we report on the biochemical properties of three so far uncharacterized members of the Arabidopsis ENT (equilibrative nucleoside transporter) family (AtENT4, AtENT6 and AtENT7). After heterologous expression in yeast, all three proteins exhibited broad substrate specificity and transported the purine nucleosides adenosine and guanosine, as well as the pyrimidine nucleosides cytidine and uridine. The apparent Km values were in the range 3–94 μM, and transport was inhibited most strongly by deoxynucleosides, and to a smaller extent by nucleobases. Typical inhibitors of mammalian ENT proteins, such as dilazep and NBMPR (nitrobenzylmercaptopurine ribonucleoside, also known as nitrobenzylthioinosine) surprisingly exerted almost no effect on Arabidopsis ENT proteins. Transport mediated by the AtENT isoforms differed in pH-dependency, e.g. AtENT7 was not affected by changes in pH, AtENT3, 4 and 6 exhibited a less pronounced pH-dependency, and AtENT1 activity was clearly pH-dependent. Using a GFP (green fluorescent protein)-fusion protein transiently expressed in tobacco leaf protoplasts, a localization of AtENT6 in the plant plasma membrane has been revealed.

Contribution of equilibrative nucleoside transporter (ENT) 2 to fluorouracil transport in rat placental trophoblast cells

2017 ◽  
Vol 32 (2) ◽  
pp. 151-156 ◽  
Author(s):  
Akinori Takagi ◽  
Tomohiro Nishimura ◽  
Tomoya Akashi ◽  
Masatoshi Tomi ◽  
Emi Nakashima
Keyword(s):  
Nucleoside Transporter ◽  
Trophoblast Cells ◽  
Equilibrative Nucleoside Transporter

Nucleoside Transporter-guided Cytarabine Conjugated Liposomes for Intracellular Methotrexate Delivery and Cooperative Choriocarcinoma Therapy

2021 ◽  
Author(s):  
Weidong Fei ◽  
Yunchun Zhao ◽  
Xiaodong Wu ◽  
Dongli Sun ◽  
Yao Yao ◽  
...  
Keyword(s):  
Targeted Delivery ◽  
Rational Design ◽  
High Efficiency ◽  
Nucleoside Transporter ◽  
Childbearing Age ◽  
Equilibrative Nucleoside Transporter ◽  
Choriocarcinoma Cells ◽  
Tumor Accumulation ◽  
Novel Strategy ◽  
Distribution Studies

Abstract The gestational trophoblastic tumor seriously endangers child productive needs and the health of women in childbearing age. Nanodrug-based therapy mediated by transporters provides novel strategy for the treatment of trophoblastic tumors. Focus on the overexpressed human equilibrative nucleoside transporter 1 (ENT1) on the membrane of choriocarcinoma cells (JEG-3), the cytarabine (Cy, a substrate of ENT1) grafted liposome (Cy-Lipo) was introduced for targeted delivery of methotrexate (Cy-Lipo@MTX) for choriocarcinoma therapy in this study. The ENT1 has high affinity for Cy-Lipo and can mediate the endocytosis of the designed nanovehicles into JEG-3 cells. The ENT1 protein maintains its transporting function through circulation and regeneration during endocytosis. Therefore, Cy-Lipo-based formulations achieved high tumor accumulation and retention in pharmacokinetic and distribution studies. More importantly, the designed Cy-lipid conjugation exhibited a synergistic therapeutic effect on choriocarcinoma. Finally, Cy-Lipo@MTX exerts an extremely powerful anti-choriocarcinoma effect with fewer side effects. This study suggests that the overexpressed ENT1 on choriocarcinoma cells holds a great potential to be a high-efficiency target for the rational design of active targeting nanotherapeutics.

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