Molecular biology and regulation of nucleoside and nucleobase transporter proteins in eukaryotes and prokaryotes

2002 ◽  
Vol 80 (5) ◽  
pp. 623-638 ◽  
Author(s):  
Miguel A Cabrita ◽  
Stephen A Baldwin ◽  
James D Young ◽  
Carol E Cass
Keyword(s):  
Molecular Biology ◽  
Vital Role ◽  
Nucleoside Transporters ◽  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Concentration Gradients ◽  
Transporter Proteins ◽  
Equilibrative Nucleoside Transporters ◽  
Nucleoside Drugs ◽  
Nucleobase Transport

The molecular cloning of cDNAs encoding nucleoside transporter proteins has greatly advanced understanding of how nucleoside permeants are translocated across cell membranes. The nucleoside transporter proteins identified thus far have been categorized into five distinct superfamilies. Two of these superfamilies, the equilibrative and concentrative nucleoside transporters, have human members and these will be examined in depth in this review. The human equilibrative nucleoside transporters translocate nucleosides and nucleobases bidirectionally down their concentration gradients and are important in the uptake of anticancer and antiviral nucleoside drugs. The human concentrative nucleoside transporters cotranslocate nucleosides and sodium unidirectionally against the nucleoside concentration gradients and play a vital role in certain tissues. The regulation of nucleoside and nucleobase transporters is being studied more intensely now that more tools are available. This review provides an overview of recent advances in the molecular biology and regulation of the nucleoside and nucleobase transporters.Key words: nucleoside transporter, nucleoside transport, nucleobase transporter, nucleobase transport, regulation of nucleoside and nucleobase transport, nucleoside drugs.

scholarly journals Current Progress on Equilibrative Nucleoside Transporter Function and Inhibitor Design

2019 ◽  
Vol 24 (10) ◽  
pp. 953-968 ◽  
Author(s):  
Shahid Rehan ◽  
Saman Shahid ◽  
Tiina A. Salminen ◽  
Veli-Pekka Jaakola ◽  
Ville O. Paavilainen
Keyword(s):  
Small Molecule ◽  
Family Members ◽  
Nucleoside Analogs ◽  
Homology Model ◽  
Nucleoside Transporters ◽  
Nucleoside Transporter ◽  
Surface Receptors ◽  
Cellular Import ◽  
Nucleoside Drugs ◽  
Small Molecule Modulators

Physiological nucleosides are used for the synthesis of DNA, RNA, and ATP in the cell and serve as universal mammalian signaling molecules that regulate physiological processes such as vasodilation and platelet aggregation by engaging with cell surface receptors. The same pathways that allow uptake of physiological nucleosides mediate the cellular import of synthetic nucleoside analogs used against cancer, HIV, and other viral diseases. Physiological nucleosides and nucleoside drugs are imported by two families of nucleoside transporters: the SLC28 concentrative nucleoside transporters (CNTs) and SLC29 equilibrative nucleoside transporters (ENTs). The four human ENT paralogs are expressed in distinct tissues, localize to different subcellular sites, and transport a variety of different molecules. Here we provide an overview of the known structure–function relationships of the ENT family with a focus on ligand binding and transport in the context of a new hENT1 homology model. We provide a generic residue numbering system for the different ENTs to facilitate the interpretation of mutational data produced using different ENT homologs. The discovery of paralog-selective small-molecule modulators is highly relevant for the design of new therapies and for uncovering the functions of poorly characterized ENT family members. Here, we discuss recent developments in the discovery of new paralog-selective small-molecule ENT inhibitors, including new natural product-inspired compounds. Recent progress in the ability to heterologously produce functional ENTs will allow us to gain insight into the structure and functions of different ENT family members as well as the rational discovery of highly selective inhibitors.

Adenosine transport: Recent advances in the molecular biology of nucleoside transporter proteins

1998 ◽  
Vol 45 (3-4) ◽  
pp. 277-287 ◽  
Author(s):  
Lori L. Jennings ◽  
Carol E. Cass ◽  
Mabel W.L. Ritzel ◽  
Sylvia Y.M. Yao ◽  
James D. Young ◽  
...  
Keyword(s):  
Molecular Biology ◽  
Nucleoside Transporter ◽  
Transporter Proteins ◽  
Recent Advances ◽  
Adenosine Transport

scholarly journals Nucleoside transport and proliferative rate in human thymocytes and lymphocytes

Blood ◽  
1989 ◽  
Vol 74 (6) ◽  
pp. 2038-2042 ◽  
Author(s):  
CL Smith ◽  
LM Pilarski ◽  
ML Egerton ◽  
JS Wiley
Keyword(s):  
Cytosine Arabinoside ◽  
Binding Sites ◽  
Flow Cytometric Analysis ◽  
Fold Increase ◽  
S Phase ◽  
Nucleoside Transporters ◽  
Labeling Index ◽  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Equilibrative Nucleoside Transporter

The thymus is a site of active T-lymphoid cell proliferation and DNA synthesis. In this study, the capacity of human thymocytes for nucleoside transport was assessed both by cytosine arabinoside influx and by equilibrium binding of nitrobenzylmercaptopurine riboside (NBMPR), a specific ligand for the equilibrative nucleoside transporter of leukocytes. The proportion of freshly isolated thymocytes synthesizing DNA was 8.6% +/- 2.1% (n = 12) by 3H-thymidine labeling index and 7.8% +/- 2.9% (n = 4) S-phase cells by flow cytometric analysis of DNA content. In comparison, both methods gave proliferation S-phase values less than 1% for peripheral blood lymphocytes (PBLs). Thymocytes expressed a high density of specific NBMPR binding sites (26,068 +/- 8,776 sites per cell, n = 12) as compared with PBLs (1,123 +/- 553 sites per cell, n = 8). The initial influx of cytosine arabinoside into thymocytes was 14-fold greater than into PBLs, and in both cell types the influx of nucleoside was totally inhibited by 0.5 mumol/L NBMPR, which is known to inhibit the major equilibrative nucleoside transporter in white blood cells. Depletion of mature CD3+ cells from the thymocyte preparation by anti-CD3 antibody left a residual population with both increased labeling index and up to twofold greater density of NBMPR binding sites. When PBLs were cultured for 48 hours with the T-cell mitogen phytohemagglutinin, a 40-fold increase in labeling index was observed, together with a 30-fold increase in the density of specific NBMPR binding sites. Thus, fresh thymocytes from human thymus are actively proliferating and express high densities of a functional nucleoside transporter. The more immature cells in the thymocyte population which are proliferating more actively have a greater density of nucleoside transporters than the whole population. In contrast, mitotically inactive PBLs-have few nucleoside transporters, but after mitogenic stimulation PBLs express large numbers of this transmembrane molecule.

Renal nucleoside transporters: physiological and clinical implicationsThis paper is one of a selection of papers published in this Special Issue, entitled CSBMCB — Membrane Proteins in Health and Disease.

2006 ◽  
Vol 84 (6) ◽  
pp. 844-858 ◽  
Author(s):  
Adam N. Elwi ◽  
Vijaya L. Damaraju ◽  
Stephen A. Baldwin ◽  
James D. Young ◽  
Michael B. Sawyer ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Membrane Proteins ◽  
Organic Cation ◽  
Nucleoside Transporters ◽  
Nucleoside Transport ◽  
Multidrug Resistance Proteins ◽  
Renal Epithelia ◽  
Renal Handling ◽  
Basolateral Membranes ◽  
Nucleoside Drugs

Renal handling of physiological and pharmacological nucleosides is a major determinant of their plasma levels and tissue availabilities. Additionally, the pharmacokinetics and normal tissue toxicities of nucleoside drugs are influenced by their handling in the kidney. Renal reabsorption or secretion of nucleosides is selective and dependent on integral membrane proteins, termed nucleoside transporters (NTs) present in renal epithelia. The 7 known human NTs (hNTs) exhibit varying permeant selectivities and are divided into 2 protein families: the solute carrier (SLC) 29 (SLC29A1, SLC29A2, SLC29A3, SLC29A4) and SLC28 (SLC28A1, SLC28A2, SLC28A3) proteins, otherwise known, respectively, as the human equilibrative NTs (hENTs, hENT1, hENT2, hENT3, hENT4) and human concentrative NTs (hCNTs, hCNT1, hCNT2, hCNT3). The well characterized hENTs (hENT1 and hENT2) are bidirectional facilitative diffusion transporters in plasma membranes; hENT3 and hENT4 are much less well known, although hENT3, found in lysosomal membranes, transports nucleosides and is pH dependent, whereas hENT4–PMAT is a H+-adenosine cotransporter as well as a monoamine–organic cation transporter. The 3 hCNTs are unidirectional secondary active Na+-nucleoside cotransporters. In renal epithelial cells, hCNT1, hCNT2, and hCNT3 at apical membranes, and hENT1 and hENT2 at basolateral membranes, apparently work in concert to mediate reabsorption of nucleosides from lumen to blood, driven by Na+ gradients. Secretion of some physiological nucleosides, therapeutic nucleoside analog drugs, and nucleotide metabolites of therapeutic nucleoside and nucleobase drugs likely occurs through various xenobiotic transporters in renal epithelia, including organic cation transporters, organic anion transporters, multidrug resistance related proteins, and multidrug resistance proteins. Mounting evidence suggests that hENT1 may have a presence at both apical and basolateral membranes of renal epithelia, and thus may participate in both selective secretory and reabsorptive fluxes of nucleosides. In this review, the renal handling of nucleosides is examined with respect to physiological and clinical implications for the regulation of human kidney NTs and adenosine signaling, intracellular nucleoside transport, and nephrotoxicities associated with some nucleoside drugs.

scholarly journals Subtype-specific regulation of equilibrative nucleoside transporters by protein kinase CK2

2005 ◽  
Vol 386 (2) ◽  
pp. 281-289 ◽  
Author(s):  
Meaghan STOLK ◽  
Elizabeth COOPER ◽  
Greg VILK ◽  
David W. LITCHFIELD ◽  
James R. HAMMOND
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Ck2 ◽  
Nucleoside Transporters ◽  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Interacting Protein ◽  
Human Osteosarcoma Cells ◽  
Specific Regulation ◽  
Membrane Lifetime ◽  
Kinase Ck2

Two subtypes of equilibrative transporters, es (equilibrative inhibitor-sensitive) and ei (equilibrative inhibitor-insensitive), are responsible for the majority of nucleoside flux across mammalian cell membranes. Sequence analyses of the representative genes, ENT1 {equilibrative nucleoside transporter 1; also known as SLC29A1 [solute carrier family 29 (nucleoside transporters), member 1]} and ENT2 (SLC29A2), suggest that protein kinase CK2-mediated phosphorylation may be involved in the regulation of es- and ei-mediated nucleoside transport. We used human osteosarcoma cells transfected with catalytically active or inactive α′ and α subunits of CK2 to assess the effects of CK2 manipulation on nucleoside transport activity. Expression of inactive CK2α′ (decreased CK2α′ activity) increased the number of binding sites (∼1.5-fold) for the es-specific probe [3H]NBMPR ([3H]nitrobenzylthioinosine), and increased (∼1.8-fold) the Vmax for 2-chloro[3H]adenosine of the NBMPR-sensitive (es) nucleoside transporter. There was a concomitant decrease in the Vmax of the NBMPR-resistant (ei-mediated) uptake of 2-chloro[3H]adenosine. This inhibition of CK2α′ activity had no effect, however, on either the KD of [3H]NBMPR binding or the Km of 2-chloro[3H]adenosine uptake. Quantitative PCR showed a transient decrease in the expression of both hENT1 (human ENT1) and hENT2 mRNAs within 4–12 h of induction of the inactive CK2α′ subunit, but both transcripts had returned to control levels by 24 h. These data suggest that inhibition of CK2α′ reduced ei activity by attenuation of hENT2 transcription, while the increase in es/hENT1 activity was mediated by post-translational action of CK2. The observed modification in es activity was probably due to a CK2α′-mediated change in the phosphorylation state of the ENT1 protein, or an interacting protein, effecting an increase in the plasma membrane lifetime of the transport proteins.

scholarly journals Interferon-γ regulates nucleoside transport systems in macrophages through signal transduction and activator of transduction factor 1 (STAT1)-dependent and -independent signalling pathways

2003 ◽  
Vol 375 (3) ◽  
pp. 777-783 ◽  
Author(s):  
Concepció SOLER ◽  
Antonio FELIPE ◽  
José GARCÍA-MANTEIGA ◽  
Maria SERRA ◽  
Elena GUILLÉN-GÓMEZ ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Signal Transduction ◽  
Interferon Γ ◽  
Nucleoside Transporters ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Transcriptional Level ◽  
Nucleoside Transporter ◽  
Protein Levels ◽  
Ifn Γ

The expressions of CNT and ENT (concentrative and equilibrative nucleoside transporters) in macrophages are differentially regulated by IFN-γ (interferon-γ). This cytokine controls gene expression through STAT1-dependent and/or -independent pathways (where STAT1 stands for signal transduction and activator of transcription 1). In the present study, the role of STAT1 in the response of nucleoside transporters to IFN-γ was studied using macrophages from STAT1 knockout mice. IFN-γ triggered an inhibition of ENT1-related nucleoside transport activity through STAT1-dependent mechanisms. Such inhibition of macrophage growth and ENT1 activity by IFN-γ is required for DNA synthesis. Interestingly, IFN-γ led to an induction of the CNT1- and CNT2-related nucleoside transport activities independent of STAT1, thus ensuring the supply of extracellular nucleosides for the STAT1-independent RNA synthesis. IFN-γ up-regulated CNT2 mRNA and CNT1 protein levels and down-regulated ENT1 mRNA in both wild-type and STAT1 knockout macrophages. This is consistent with a STAT1-independent, long-term-mediated, probably transcription-dependent, regulation of nucleoside transporter genes. Moreover, STAT1-dependent post-transcriptional mechanisms are implicated in the regulation of ENT1 activity. Although nitric oxide is involved in the regulation of ENT1 activity in B-cells at a post-transcriptional level, our results show that STAT1-dependent induction of nitric oxide by IFN-γ is not implicated in the regulation of ENT1 activity in macrophages. Our results indicate that both STAT1-dependent and -independent pathways are involved in the regulation of nucleoside transporters by IFN-γ in macrophages.

scholarly journals Functional and Molecular Characterization of Nucleobase Transport by Recombinant Human and Rat Equilibrative Nucleoside Transporters 1 and 2

2002 ◽  
Vol 277 (28) ◽  
pp. 24938-24948 ◽  
Author(s):  
Sylvia Y. M. Yao ◽  
Amy M. L. Ng ◽  
Mark F. Vickers ◽  
Manickavasagam Sundaram ◽  
Carol E. Cass ◽  
...  
Keyword(s):  
Molecular Characterization ◽  
Nucleoside Transporters ◽  
Equilibrative Nucleoside Transporters ◽  
Nucleobase Transport

scholarly journals [3H]dipyridamole binding to nucleoside transporters from guinea-pig and rat lung

1986 ◽  
Vol 240 (3) ◽  
pp. 879-883 ◽  
Author(s):  
M M Shi ◽  
J D Young
Keyword(s):  
Guinea Pig ◽  
Nucleoside Transporters ◽  
Nucleoside Transport ◽  
Affinity Binding ◽  
Nucleoside Transporter ◽  
High Affinity Binding ◽  
High Affinity ◽  
Nucleoside Transport Inhibitors ◽  
Transport Inhibitors ◽  
Low Sensitivity

Membranes from guinea-pig lung exhibited high-affinity binding of [3H]dipyridamole, a potent inhibitor of nucleoside transport. Binding (apparent KD 2 nM) was inhibited by the nucleoside-transport inhibitors nitrobenzylthioinosine (NBMPR), dilazep and lidoflazine and by the transported nucleosides uridine and adenosine. In contrast, there was no detectable high-affinity binding of [3H]dipyridamole to lung membranes from the rat, a species whose nucleoside transporters exhibit a low sensitivity to dipyridamole inhibition. Bmax. values for high-affinity binding of [3H]dipyridamole and [3H]NBMPR to guinea-pig membranes were similar, suggesting that these structurally unrelated ligands bind to the NBMPR-sensitive nucleoside transporter with the same stoichiometry.

scholarly journals Cysteine-accessibility analysis of transmembrane domains 11–13 of human concentrative nucleoside transporter 3

2006 ◽  
Vol 394 (2) ◽  
pp. 389-398 ◽  
Author(s):  
Jing Zhang ◽  
Tracey Tackaberry ◽  
Mabel W. L. Ritzel ◽  
Taylor Raborn ◽  
Gerry Barron ◽  
...  
Keyword(s):  
Expression System ◽  
Transmembrane Domains ◽  
Nucleoside Transport ◽  
Cysteine Residues ◽  
Nucleoside Transporter ◽  
Yeast Expression System ◽  
Cysteine Substitution ◽  
Concentrative Nucleoside Transporter ◽  
Translocation Pathway ◽  
Nucleoside Drugs

hCNT3 (human concentrative nucleoside transporter 3) is a nucleoside–sodium symporter that transports a broad range of naturally occurring purine and pyrimidine nucleosides as well as anticancer nucleoside drugs. To understand its uridine binding and translocation mechanisms, a cysteine-less version of hCNT3 was constructed and used for cysteine-accessibility and permeant-protection assays. Cysteine-less hCNT3, with 14 endogenous cysteine residues changed to serine, displayed wild-type properties in a yeast expression system, indicating that endogenous cysteine residues are not essential for hCNT3-mediated nucleoside transport. A series of cysteine-substitution mutants spanning predicted TMs (transmembrane domains) 11–13 was constructed and tested for accessibility to thiol-specific reagents. Mutants M496C, G498C, F563C, A594C, G598C and A606C had no detectable transport activity, indicating that a cysteine substitution at each of these positions was not tolerated. Two functional mutants in putative TM 11 (L480C and S487C) and four in putative TM 12 (N565C, T557C, G567C and I571C) were partially inhibited by MTS (methanethiosulphonate) reagent and high concentrations of uridine protected against inhibition, indicating that TMs 11 and 12 may form part of the nucleoside translocation pathway. The lack of accessibility of MTS reagents to TM 13 mutants suggests that TM 13 is not exposed to the nucleoside translocation pathway. Furthermore, G567C, N565C and I571C mutants were only sensitive to MTSEA (MTS-ethylammonium), a membranepermeant thiol reagent, indicating that these residues may be accessible from the cytoplasmic side of the membrane, providing evidence in support of the predicted orientation of TM 12 in the current putative topology model of hCNT3.

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