Recent advances in studies on biochemical and structural properties of equilibrative and concentrative nucleoside transporters.

Nucleoside transporters (NT) facilitate the movement of nucleosides and nucleobases across cell membranes. NT-mediated transport is vital for the synthesis of nucleic acids in cells that lack de novo purine synthesis. Some nucleosides display biological activity and act as signalling molecules. For example, adenosine exerts a potent action on many physiological processes including vasodilatation, hormone and neurotransmitter release, platelet aggregation, and lipolysis. Therefore, carrier-mediated transport of this nucleoside plays an important role in modulating cell function, because the efficiency of the transport processes determines adenosine availability to its receptors or to metabolizing enzymes. Nucleoside transporters are also key elements in anticancer and antiviral therapy with the use of nucleoside analogues. Mammalian cells possess two major nucleoside transporter families: equilibrative (ENT) and concentrative (CNT) Na(+)-dependent ones. This review characterizes gene loci, substrate specificity, tissue distribution, membrane topology and structure of ENT and CNT proteins. Regulation of nucleoside transporters by various factors is also presented.

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Equilibrative Nucleoside Transporters Mediate the Import of Nicotinamide Riboside and Nicotinic Acid Riboside into Human Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22031391 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1391

Author(s):

Andrey Kropotov ◽

Veronika Kulikova ◽

Kirill Nerinovski ◽

Alexander Yakimov ◽

Maria Svetlova ◽

...

Keyword(s):

Nicotinic Acid ◽

Mammalian Cells ◽

Experimental Models ◽

The Body ◽

Nucleoside Transporters ◽

Hek293 Cells ◽

Nucleoside Transporter ◽

Vitamin B3 ◽

Animal Cells ◽

Nicotinamide Riboside

Nicotinamide riboside (NR), a new form of vitamin B3, is an effective precursor of nicotinamide adenine dinucleotide (NAD+) in human and animal cells. The introduction of NR into the body effectively increases the level of intracellular NAD+ and thereby restores physiological functions that are weakened or lost in experimental models of aging and various pathologies. Despite the active use of NR in applied biomedicine, the mechanism of its transport into mammalian cells is currently not understood. In this study, we used overexpression of proteins in HEK293 cells, and metabolite detection by NMR, to show that extracellular NR can be imported into cells by members of the equilibrative nucleoside transporter (ENT) family ENT1, ENT2, and ENT4. After being imported into cells, NR is readily metabolized resulting in Nam generation. Moreover, the same ENT-dependent mechanism can be used to import the deamidated form of NR, nicotinic acid riboside (NAR). However, NAR uptake into HEK293 cells required the stimulation of its active utilization in the cytosol such as phosphorylation by NR kinase. On the other hand, we did not detect any NR uptake mediated by the concentrative nucleoside transporters (CNT) CNT1, CNT2, or CNT3, while overexpression of CNT3, but not CNT1 or CNT2, moderately stimulated NAR utilization by HEK293 cells.

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Functional characterization of a recombinant sodium-dependent nucleoside transporter with selectivity for pyrimidine nucleosides (cNT1rat) by transient expression in cultured mammalian cells

Biochemical Journal ◽

10.1042/bj3170457 ◽

1996 ◽

Vol 317 (2) ◽

pp. 457-465 ◽

Cited By ~ 49

Author(s):

Xiao FANG ◽

Fiona E. PARKINSON ◽

Delores A. MOWLES ◽

James D. YOUNG ◽

Carol E. CASS

Keyword(s):

Mammalian Cells ◽

Expression System ◽

Functional Characterization ◽

Transport Processes ◽

Nucleoside Transport ◽

Single Type ◽

Nucleoside Transporter ◽

Pyrimidine Nucleosides ◽

Transporter Protein ◽

Sodium Dependent

We have demonstrated that monkey kidney (COS-1) cells have a single type of nucleoside transport process, which, because it was equilibrative, sodium-independent and could be inhibited by nitrobenzylthioinosine (NBMPR), was identified as the ‘equilibrative sensitive’ or ‘es’ transporter. Using NBMPR or dilazep to inhibit the endogenous nucleoside transport activity, we have transiently expressed a cDNA that encodes an inhibitor-insensitive, concentrative nucleoside transporter protein (cNT1rat) of rat intestine in COS-1 cells. The production of recombinant cNT1rat was examined by immunoblotting using an epitope-tagged construct and by analysis of inward fluxes of 3H-labelled nucleosides. Recombinant cNT1rat was sodium-dependent and selective for pyrimidine nucleosides, with approximate Km values of 21 μM, 12.5 μM and 15 μM for uridine, thymidine and adenosine, respectively. Although adenosine exhibited high affinity for the recombinant transporter, its Vmax value was low. A variety of anti-viral and anti-cancer nucleoside drugs inhibited cNT1rat-mediated uptake of uridine by transfected COS-1 cells although to different extents (Floxidine > Idoxuridine > Zidovudine > Zalcitabine > Cytarabine > Gemcitabine), suggesting that the concentrative pyrimidine-selective nucleoside transporters, of which cNT1rat is a representative, may play a role in cellular uptake of these drugs. The cNT1rat/COS-1 expression system is a useful tool for analysis of cNT1rat-mediated transport processes.

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Uptake of Nitrobenzylthioinosine and Purine β-l-Nucleosides by Intracellular Toxoplasma gondii

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.10.3247-3251.2003 ◽

2003 ◽

Vol 47 (10) ◽

pp. 3247-3251 ◽

Cited By ~ 23

Author(s):

Omar N. Al Safarjalani ◽

Fardos N. M. Naguib ◽

Mahmoud H. el Kouni

Keyword(s):

Toxoplasma Gondii ◽

Mammalian Cells ◽

Nucleoside Transporters ◽

Host Cells ◽

Purine Nucleoside ◽

Nucleoside Transport ◽

Fibroblast Cells ◽

Nucleoside Transporter ◽

Intracellular Parasites ◽

Infected Cells

ABSTRACT Intracellular Toxoplasma gondii grown in human foreskin fibroblast cells transported nitrobenzylthioinosine {NBMPR; 6-[(4-nitrobenzyl)mercapto]-9-β-d-ribofuranosylpurine}, an inhibitor of nucleoside transport in mammalian cells, as well as the nonphysiological β-l-enantiomers of purine nucleosides, β-l-adenosine, β-l-deoxyadenosine, and β-l-guanosine. The β-l-pyrimidine nucleosides, β-l-uridine, β-l-cytidine, and β-l-thymidine, were not transported. The uptake of NBMPR and the nonphysiological purine nucleoside β-l-enantiomers by the intracellular parasites also implies that Toxoplasma-infected cells can transport these nucleosides. In sharp contrast, under the same conditions, uninfected fibroblast cells did not transport NBMPR or any of the unnatural β-l-nucleosides. β-d-Adenosine and dipyridamole, another inhibitor of nucleoside transport, inhibited the uptake of NBMPR and β-l-stereoisomers of the purine nucleosides by intracellular Toxoplasma and Toxoplasma-infected cells. Furthermore, infection with a Toxoplasma mutant deficient in parasite adenosine/purine nucleoside transport reduced or abolished the uptake of β-d-adenosine, NBMPR, and purine β-l-nucleosides. Hence, the presence of the Toxoplasma adenosine/purine nucleoside transporters is apparently essential for the uptake of NBMPR and purine β-l-nucleosides by intracellular Toxoplasma and Toxoplasma-infected cells. These results also demonstrate that, in contrast to the mammalian nucleoside transporters, the Toxoplasma adenosine/purine nucleoside transporter(s) lacks stereospecificity and substrate specificity in the transport of purine nucleosides. In addition, infection with T. gondii confers the properties of the parasite's purine nucleoside transport on the parasitized host cells and enables the infected cells to transport purine nucleosides that were not transported by uninfected cells. These unique characteristics of purine nucleoside transport in T. gondii may aid in the identification of new promising antitoxoplasmic drugs.

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Differential Subcellular Distribution of Cytokinins: How Does Membrane Transport Fit into the Big Picture?

International Journal of Molecular Sciences ◽

10.3390/ijms22073428 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3428

Author(s):

Daniel Nedvěd ◽

Petr Hošek ◽

Petr Klíma ◽

Klára Hoyerová

Keyword(s):

Membrane Transport ◽

Nucleoside Transporters ◽

Long Distance ◽

Cytokinin Biosynthesis ◽

Signalling Molecules ◽

Equilibrative Nucleoside Transporters ◽

Physiological Processes ◽

Big Picture ◽

Membrane Bound ◽

Related Compounds

Cytokinins are a class of phytohormones, signalling molecules specific to plants. They act as regulators of diverse physiological processes in complex signalling pathways. It is necessary for plants to continuously regulate cytokinin distribution among different organs, tissues, cells, and compartments. Such regulatory mechanisms include cytokinin biosynthesis, metabolic conversions and degradation, as well as cytokinin membrane transport. In our review, we aim to provide a thorough picture of the latter. We begin by summarizing cytokinin structures and physicochemical properties. Then, we revise the elementary thermodynamic and kinetic aspects of cytokinin membrane transport. Next, we review which membrane-bound carrier proteins and protein families recognize cytokinins as their substrates. Namely, we discuss the families of “equilibrative nucleoside transporters” and “purine permeases”, which translocate diverse purine-related compounds, and proteins AtPUP14, AtABCG14, AtAZG1, and AtAZG2, which are specific to cytokinins. We also address long-distance cytokinin transport. Putting all these pieces together, we finally discuss cytokinin distribution as a net result of these processes, diverse in their physicochemical nature but acting together to promote plant fitness.

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Recent advances in the molecular biology of nucleoside transporters of mammalian cells

Biochemistry and Cell Biology ◽

10.1139/o98-095 ◽

1998 ◽

Vol 76 (5) ◽

pp. 761-770 ◽

Cited By ~ 102

Author(s):

Carol E Cass ◽

James D Young ◽

Stephen A Baldwin

Keyword(s):

Molecular Biology ◽

Mammalian Cells ◽

Cell Types ◽

Functional Expression ◽

Transport Proteins ◽

Transport Processes ◽

Nucleoside Transport ◽

Nucleoside Transporter ◽

Protein Families ◽

Public Data

Nucleosides are hydrophilic molecules and require specialized transport proteins for permeation of cell membranes. There are two types of nucleoside transport processes: equilibrative bidirectional processes driven by chemical gradients and inwardly directed concentrative processes driven by the sodium electrochemical gradient. The equilibrative nucleoside transport processes (es, ei) are found in most mammalian cell types, whereas the concentrative nucleoside transport processes (cit, cif, cib, csg, cs) are present primarily in specialized epithelia. Using a variety of cloning strategies and functional expression in oocytes of Xenopus laevis, we have isolated and characterized cDNAs encoding the rat and human nucleoside transporter proteins of the four major nucleoside transport processes of mammalian cells (es, ei, cit, cif). From the sequence relationships of these proteins with each other and with sequences in the public data bases, we have concluded that the equilibrative and concentrative nucleoside transport processes are mediated by members of two previously unrecognized groups of integral membrane proteins, which we have designated the equilibrative nucleoside transporter (ENT) and the concentrative nucleoside transporter (CNT) protein families. This review summarizes the current state of knowledge in the molecular biology of the ENT and CNT protein families, focusing on the characteristics of the four human (h) and rat (r) nucleoside transport proteins (r/hENT1, r/hENT2, r/hCNT1, r/hCNT2).Key words: nucleoside transporter, equilibrative, concentrative, ENT, CNT.

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Allosteric and transport modulation of human concentrative nucleoside transporter 3 at the atomic scale

Physical Chemistry Chemical Physics ◽

10.1039/d1cp03756k ◽

2021 ◽

Author(s):

Huaichuan Duan ◽

Zhou Yanxia ◽

XiaoDong Shi ◽

Qing Luo ◽

Gao Jiaxing ◽

...

Keyword(s):

Nucleoside Transporters ◽

Atomic Scale ◽

Nucleoside Transport ◽

Transmembrane Transport ◽

Nucleoside Transporter ◽

Nucleotide Synthesis ◽

Physiological Processes ◽

Concentrative Nucleoside Transporter ◽

Transport Modulation ◽

In Cells

Nucleosides are important precursors of nucleotide synthesis in cells, and nucleoside transporters play an important role in many physiological processes by mediating its transmembrane transport and absorption. During nucleoside transport,...

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Set1 Targets Genes with Essential Identity and Tumor-Suppressing Functions in Planarian Stem Cells

Genes ◽

10.3390/genes12081182 ◽

2021 ◽

Vol 12 (8) ◽

pp. 1182

Author(s):

Prince Verma ◽

Court K. M. Waterbury ◽

Elizabeth M. Duncan

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Mammalian Cells ◽

Cell Function ◽

Genotoxic Stress ◽

Specific Gene ◽

Cell Identity ◽

Chromatin Signature ◽

Lysine Methyltransferase

Tumor suppressor genes (TSGs) are essential for normal cellular function in multicellular organisms, but many TSGs and tumor-suppressing mechanisms remain unknown. Planarian flatworms exhibit particularly robust tumor suppression, yet the specific mechanisms underlying this trait remain unclear. Here, we analyze histone H3 lysine 4 trimethylation (H3K4me3) signal across the planarian genome to determine if the broad H3K4me3 chromatin signature that marks essential cell identity genes and TSGs in mammalian cells is conserved in this valuable model of in vivo stem cell function. We find that this signature is indeed conserved on the planarian genome and that the lysine methyltransferase Set1 is largely responsible for creating it at both cell identity and putative TSG loci. In addition, we show that depletion of set1 in planarians induces stem cell phenotypes that suggest loss of TSG function, including hyperproliferation and an abnormal DNA damage response (DDR). Importantly, this work establishes that Set1 targets specific gene loci in planarian stem cells and marks them with a conserved chromatin signature. Moreover, our data strongly suggest that Set1 activity at these genes has important functional consequences both during normal homeostasis and in response to genotoxic stress.

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CALINCA—A Novel Pipeline for the Identification of lncRNAs in Podocyte Disease

Cells ◽

10.3390/cells10030692 ◽

2021 ◽

Vol 10 (3) ◽

pp. 692

Author(s):

Sweta Talyan ◽

Samantha Filipów ◽

Michael Ignarski ◽

Magdalena Smieszek ◽

He Chen ◽

...

Keyword(s):

Cell Biology ◽

Mammalian Cells ◽

De Novo ◽

Depth Information ◽

Gene Products ◽

Classical Analysis ◽

Protein Coding ◽

Bioinformatic Pipeline ◽

Non Coding Rnas ◽

Filtration Unit

Diseases of the renal filtration unit—the glomerulus—are the most common cause of chronic kidney disease. Podocytes are the pivotal cell type for the function of this filter and focal-segmental glomerulosclerosis (FSGS) is a classic example of a podocytopathy leading to proteinuria and glomerular scarring. Currently, no targeted treatment of FSGS is available. This lack of therapeutic strategies is explained by a limited understanding of the defects in podocyte cell biology leading to FSGS. To date, most studies in the field have focused on protein-coding genes and their gene products. However, more than 80% of all transcripts produced by mammalian cells are actually non-coding. Here, long non-coding RNAs (lncRNAs) are a relatively novel class of transcripts and have not been systematically studied in FSGS to date. The appropriate tools to facilitate lncRNA research for the renal scientific community are urgently required due to a row of challenges compared to classical analysis pipelines optimized for coding RNA expression analysis. Here, we present the bioinformatic pipeline CALINCA as a solution for this problem. CALINCA automatically analyzes datasets from murine FSGS models and quantifies both annotated and de novo assembled lncRNAs. In addition, the tool provides in-depth information on podocyte specificity of these lncRNAs, as well as evolutionary conservation and expression in human datasets making this pipeline a crucial basis to lncRNA studies in FSGS.

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A Narrative Review on the Role of AMPK on De Novo Lipogenesis in Non-Alcoholic Fatty Liver Disease: Evidence from Human Studies

Cells ◽

10.3390/cells10071822 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1822

Author(s):

Christian von Loeffelholz ◽

Sina M. Coldewey ◽

Andreas L. Birkenfeld

Keyword(s):

Liver Disease ◽

Fatty Liver ◽

Fatty Liver Disease ◽

Mammalian Cells ◽

De Novo ◽

Adenosine Monophosphate ◽

De Novo Lipogenesis ◽

Alcoholic Fatty Liver ◽

Alcoholic Fatty Liver Disease

5′AMP-activated protein kinase (AMPK) is known as metabolic sensor in mammalian cells that becomes activated by an increasing adenosine monophosphate (AMP)/adenosine triphosphate (ATP) ratio. The heterotrimeric AMPK protein comprises three subunits, each of which has multiple phosphorylation sites, playing an important role in the regulation of essential molecular pathways. By phosphorylation of downstream proteins and modulation of gene transcription AMPK functions as a master switch of energy homeostasis in tissues with high metabolic turnover, such as the liver, skeletal muscle, and adipose tissue. Regulation of AMPK under conditions of chronic caloric oversupply emerged as substantial research target to get deeper insight into the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Evidence supporting the role of AMPK in NAFLD is mainly derived from preclinical cell culture and animal studies. Dysbalanced de novo lipogenesis has been identified as one of the key processes in NAFLD pathogenesis. Thus, the scope of this review is to provide an integrative overview of evidence, in particular from clinical studies and human samples, on the role of AMPK in the regulation of primarily de novo lipogenesis in human NAFLD.

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Genetic analysis of intracellular aminoglycerophospholipid traffic

Biochemistry and Cell Biology ◽

10.1139/o03-075 ◽

2004 ◽

Vol 82 (1) ◽

pp. 156-169 ◽

Cited By ~ 19

Author(s):

Dennis R Voelker

Keyword(s):

Genetic Analysis ◽

Mammalian Cells ◽

Molecular Genetic ◽

Family Members ◽

Molecular Genetic Analysis ◽

Transport Processes ◽

Membrane Biogenesis ◽

Independent Manner ◽

Multiple Genes ◽

Vacuolar Protein

Inter- and intramembrane phospholipid transport processes are central features of membrane biogenesis and homeostasis. Relatively recent successes in the molecular genetic analysis of aminoglycerophospholipid transport processes in both yeast and mammalian cells are now providing important new information defining specific protein and lipid components that participate in these reactions. Studies focused on phosphatidylserine (PtdSer) transport to the mitochondria reveal that the process is regulated by ubiquitination. In addition, a specific mutation disrupts PtdSer transport between mitochondrial membranes. Analysis of PtdSer transport from the endoplasmic reticulum to the locus of PtdSer decarboxylase 2 demonstrates the requirement for a phosphatidylinositol-4-kinase, a phosphatidylinositol-binding protein, and the C2 domain of the decarboxylase. Examination of NBD-phosphatidylcholine transport demonstrates the involvement of the prevacuolar compartment and a requirement for multiple genes involved in regulating vacuolar protein sorting for transport of the lipid to the vacuole. In intramembrane transport, multiple genes are now identified including those encoding multidrug resistant protein family members, DNF family members, ATP binding cassette transporters, and pleiotropic drug resistance family members. The scramblase family constitutes a collection of putative transmembrane transporters that function in an ATP-independent manner. The genetic analysis of lipid traffic is uncovering new molecules involved in all aspects of the regulation and execution of the transport steps and also providing essential tools to critically test the involvement of numerous candidate molecules.Key words: lipid transport, lipid sorting, membrane biogenesis, organelles, flippase.

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