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Marçal Pastor-anglada - One of the best experts on this subject based on the ideXlab platform.
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MOL #63552 1
2016Co-Authors: Ekaitz Errasti-murugarren, Javier F Casado, Miriam Molina-arcas, Marçal Pastor-angladaAbstract:The human Concentrative Nucleoside Transporter-3 Cys602Arg (hCNT3C602R) variant shows impaired sorting to lipid rafts and altered specificity for Nucleoside-derived drug
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Galectin-4 interacts with the drug Transporter human Concentrative Nucleoside Transporter 3 to regulate its function
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2015Co-Authors: Paula Fernández-calotti, Olga Casulleras, Maria Antolin, Francisco Guarner, Marçal Pastor-angladaAbstract:The intracellular N-terminal domain of the Nucleoside and drug Transporter human Concentrative Nucleoside Transporter (hCNT)3 was used as bait in a glutathione S-transferase pull-down approach, to identify hCNT3 protein partners, using human colon homogenates as a prey source. Galectin (Gal)-4 was identified as a potential hCNT3 partner in the colon. The biochemical validation of the Gal-4-hCNT3 interaction was verified by targeted pull-down assays and coimmunoprecipitation experiments in HT-29 cells, which endogenously express hCNT3 and Gal-4. Furthermore, Gal-4 was shown to colocalize with hCNT3 in HT-29 cells. The biologic significance of this interaction was obtained from experiments in which Gal-4 was knocked down, showing that this protein is a regulator of hCNT3 trafficking and retention at the cell membrane, reducing its plasma membrane location by 70%. Conversely, the addition of Gal-4 increased hCNT3 location at the plasma membrane by 77%, thereby demonstrating that this lectin modulates hCNT3 function in colonic cells. The integrity of this partnership may be clinically relevant, because hCNT3 may be responsible for the translocation of thiopurines, such as 6-mercaptopurine, a front-line treatment in inflammatory bowel disease. The expression of Gal-4 and hCNT3 proteins is not impaired in inflamed colon from patients with Crohn's disease, thereby anticipating the integrity of this system for drug targeting.
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rCNT2 extracellular cysteines, Cys615 and Cys649, are important for maturation and sorting to the plasma membrane
FEBS letters, 2014Co-Authors: Itziar Pinilla-macua, Ana Claudio-montero, Marçal Pastor-angladaAbstract:rCNT2 is a purine-preferring Concentrative Nucleoside Transporter implicated in the regulation of extracellular adenosine levels and purinergic signaling. This study addressed the analysis of the CNT2 C-terminus tail as a domain likely to be implicated in Transporter sorting. The topological mapping of this segment revealed that Cys615 and Cys649 are important residues for the proper trafficking of CNT2 to the plasma membrane. The inhibition of protein disulfide isomerase (PDI) and ER glycosidase I and II impaired rCNT2 trafficking to the cell surface, similarly to Cys615 and Cys649 mutants. The present work suggests these two cysteine residues are relevant for the proper sorting of the Transporter and its functional performance.
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Concentrative Nucleoside Transporter 1 (hCNT1) promotes phenotypic changes relevant to tumor biology in a translocation-independent manner
Cell death & disease, 2013Co-Authors: Sandra Pérez-torras, I Huber-ruano, A. Vidal-pla, P Cano-soldado, Adela Mazo, Marçal Pastor-angladaAbstract:Nucleoside Transporters (NTs) mediate the uptake of Nucleosides and nucleobases across the plasma membrane, mostly for salvage purposes. The canonical NTs belong to two gene families, SLC29 and SLC28. The former encode equilibrative Nucleoside Transporter proteins (ENTs), which mediate the facilitative diffusion of natural Nucleosides with broad selectivity, whereas the latter encode Concentrative Nucleoside Transporters (CNTs), which are sodium-coupled and show high affinity for substrates with variable selectivity. These proteins are expressed in most cell types, exhibiting apparent functional redundancy. This might indicate that CNTs have specific roles in the physiology of the cell beyond Nucleoside salvage. Here, we addressed this possibility using adenoviral vectors to restore tumor cell expression of hCNT1 or a polymorphic variant (hCNT1S546P) lacking Nucleoside translocation ability. We found that hCNT1 restoration in pancreatic cancer cells significantly altered cell-cycle progression and phosphorylation status of key signal-transducing kinases, promoted poly-(ADP-ribose) polymerase hyperactivation and cell death and reduced cell migration. Importantly, the translocation-defective Transporter triggered these same effects on cell physiology. Moreover, this study also shows that restoration of hCNT1 expression is able to reduce tumor growth in a mouse model of pancreatic adenocarcinoma. These data predict a novel role for a NT protein, hCNT1, which appears to be independent of its role as mediator of Nucleoside uptake by cells. Thereby, hCNT1 fits the profile of a transceptor in a substrate translocation-independent manner and is likely to be relevant to tumor biology.
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Role of the Transporter regulator protein (RS1) in the modulation of Concentrative Nucleoside Transporters (CNTs) in epithelia
Molecular pharmacology, 2012Co-Authors: Ekaitz Errasti-murugarren, Paula Fernández-calotti, Itziar Pinilla-macua, Mayke Veyhl-wichmann, Maximilian Diepold, Sandra Pérez-torras, Helmut Kipp, Hermann Koepsell, Marçal Pastor-angladaAbstract:SLC28 genes encode three plasma membrane Transporter proteins, human Concentrative Nucleoside Transporter (CNT)1, CNT2, and CNT3, all of which are implicated in the uptake of natural Nucleosides and a variety of Nucleoside analogs used in the chemotherapy of cancer and viral and inflammatory diseases. Mechanisms determining their trafficking toward the plasma membrane are not well known, although this might eventually become a target for therapeutic intervention. The Transporter regulator RS1, which was initially identified as a short-term, post-transcriptional regulator of the high-affinity, Na+-coupled, glucose Transporter sodium-dependent glucose coTransporter 1, was evaluated in this study as a candidate for coordinate regulation of membrane insertion of human CNT-type proteins. With a combination of studies with mammalian cells, Xenopus laevis oocytes, and RS1-null mice, evidence that RS1 down-regulates the localization and activity at the plasma membrane of the three members of this protein family (CNT1, CNT2, and CNT3) is provided, which indicates the biochemical basis for coordinate regulation of Nucleoside uptake ability in epithelia and probably in other RS1-expressing cell types.
James D Young - One of the best experts on this subject based on the ideXlab platform.
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substituted cysteine accessibility method scam analysis of the transport domain of human Concentrative Nucleoside Transporter 3 hcnt3 and other family members reveals features of structural and functional importance
Journal of Biological Chemistry, 2017Co-Authors: Ras Mulinta, Carol E Cass, Amy M L Ng, James D YoungAbstract:Abstract The human SLC28 family of Concentrative Nucleoside Transporter (CNT) proteins has three members: hCNT1, hCNT2, and hCNT3. Na+-coupled hCNT1 and hCNT2 transport pyrimidine and purine Nucleosides, respectively, whereas hCNT3 transports both pyrimidine and purine Nucleosides utilizing Na+ and/or H+ electrochemical gradients. Escherichia coli CNT family member NupC resembles hCNT1 in permeant selectivity but is H+-coupled. Using heterologous expression in Xenopus oocytes and the engineered cysteine-less hCNT3 protein hCNT3(C−), substituted cysteine accessibility method analysis with the membrane-impermeant thiol reactive reagent p-chloromercuribenzene sulfonate was performed on the transport domain (interfacial helix 2, hairpin 1, putative transmembrane domain (TM) 7, and TM8), as well as TM9 of the scaffold domain of the protein. This systematic scan of the entire C-terminal half of hCNT3(C−) together with parallel studies of the transport domain of wild-type hCNT1 and the corresponding TMs of cysteine-less NupC(C−) yielded results that validate the newly developed structural homology model of CNT membrane architecture for human CNTs, revealed extended conformationally mobile regions within transport-domain TMs, identified pore-lining residues of functional importance, and provided evidence of an emerging novel elevator-type mechanism of Transporter function.
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Human Concentrative Nucleoside Transporter 3 Transfection with Ultrasound and Microbubbles in Nucleoside Transport Deficient HEK293 Cells Greatly
2016Co-Authors: Increases Gemcitabine Uptake, James D Young, Carol E Cass, Sylvia Y. M. Yao, Robert J. Paproski, Nicole Favis, David Evans, Roger J. ZempAbstract:Gemcitabine is a hydrophilic clinical anticancer drug that requires Nucleoside Transporters to cross plasma membranes and enter cells. Pancreatic adenocarcinomas with low levels of Nucleoside Transporters are generally resistant to gemcitabine and are currently a clinical problem. We tested whether transfection of human Concentrative Nucleoside Transporter 3 (hCNT3) using ultrasound and lipid stabilized microbubbles could increase gemcitabine uptake and sensitivity in HEK293 cells made Nucleoside transport deficient by pharmacologic treatment with dilazep. To our knowledge, no published data exists regarding the utility of using hCNT3 as a therapeutic gene to reverse gemcitabine resistance. Our ultrasound transfection system- capable of transfection of cell cultures, mouse muscle and xenograft CEM/araC tumors- increased hCNT3 mRNA and 3H-gemcitabine uptake by.2,000 – and 3,400–fold, respectively, in dilazep-treated HEK293 cells. Interestingly, HEK293 cells with both functional human equilibrative Nucleoside Transporters and hCNT3 displayed 5 % of 3H-gemcitabine uptake observed in cells with only functional hCNT3, suggesting that equilibrative Nucleoside Transporters caused significant efflux of 3H-gemcitabine. Efflux assays confirmed that dilazep could inhibit the majority of 3H-gemcitabine efflux from HEK293 cells, suggesting that hENTs were responsible for the majority of efflux from the tested cells. Oocyte uptake transport assays were also performed and provided support for our hypothesis. Gemcitabine uptake and efflux assays were also performed on pancreatic cancer AsPC-1 and MIA PaCa-2 cells with similar results to that o
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human Concentrative Nucleoside Transporter 3 transfection with ultrasound and microbubbles in Nucleoside transport deficient hek293 cells greatly increases gemcitabine uptake
PLOS ONE, 2013Co-Authors: Robert J. Paproski, James D Young, Carol E Cass, Nicole Favis, David H Evans, Roger J. ZempAbstract:Gemcitabine is a hydrophilic clinical anticancer drug that requires Nucleoside Transporters to cross plasma membranes and enter cells. Pancreatic adenocarcinomas with low levels of Nucleoside Transporters are generally resistant to gemcitabine and are currently a clinical problem. We tested whether transfection of human Concentrative Nucleoside Transporter 3 (hCNT3) using ultrasound and lipid stabilized microbubbles could increase gemcitabine uptake and sensitivity in HEK293 cells made Nucleoside transport deficient by pharmacologic treatment with dilazep. To our knowledge, no published data exists regarding the utility of using hCNT3 as a therapeutic gene to reverse gemcitabine resistance. Our ultrasound transfection system - capable of transfection of cell cultures, mouse muscle and xenograft CEM/araC tumors - increased hCNT3 mRNA and 3H-gemcitabine uptake by >2,000– and 3,400–fold, respectively, in dilazep-treated HEK293 cells. Interestingly, HEK293 cells with both functional human equilibrative Nucleoside Transporters and hCNT3 displayed 5% of 3H-gemcitabine uptake observed in cells with only functional hCNT3, suggesting that equilibrative Nucleoside Transporters caused significant efflux of 3H-gemcitabine. Efflux assays confirmed that dilazep could inhibit the majority of 3H-gemcitabine efflux from HEK293 cells, suggesting that hENTs were responsible for the majority of efflux from the tested cells. Oocyte uptake transport assays were also performed and provided support for our hypothesis. Gemcitabine uptake and efflux assays were also performed on pancreatic cancer AsPC-1 and MIA PaCa-2 cells with similar results to that of HEK293 cells. Using the MTS proliferation assay, dilazep-treated HEK293 cells demonstrated 13-fold greater resistance to gemcitabine compared to dilazep-untreated HEK293 cells and this resistance could be reversed by transfection of hCNT3 cDNA. We propose that transfection of hCNT3 cDNA using ultrasound and microbubbles may be a method to reverse gemcitabine resistance in pancreatic tumors that have little Nucleoside transport activity which are resistant to almost all current anticancer therapies.
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conserved glutamate residues glu 343 and glu 519 provide mechanistic insights into cation Nucleoside cotransport by human Concentrative Nucleoside Transporter hcnt3
Journal of Biological Chemistry, 2009Co-Authors: Melissa D Slugoski, Carol E Cass, Stephen A. Baldwin, Kyla M Smith, Sylvia Y. M. Yao, Edward Karpinski, James D YoungAbstract:Human Concentrative Nucleoside Transporter 3 (hCNT3) utilizes electrochemical gradients of both Na+ and H+ to accumulate pyrimidine and purine Nucleosides within cells. We have employed radioisotope flux and electrophysiological techniques in combination with site-directed mutagenesis and heterologous expression in Xenopus oocytes to identify two conserved pore-lining glutamate residues (Glu-343 and Glu-519) with essential roles in hCNT3 Na+/Nucleoside and H+/Nucleoside cotransport. Mutation of Glu-343 and Glu-519 to aspartate, glutamine, and cysteine severely compromised hCNT3 transport function, and changes included altered Nucleoside and cation activation kinetics (all mutants), loss or impairment of H+ dependence (all mutants), shift in Na+:Nucleoside stoichiometry from 2:1 to 1:1 (E519C), complete loss of catalytic activity (E519Q) and, similar to the corresponding mutant in Na+-specific hCNT1, uncoupled Na+ currents (E343Q). Consistent with close-proximity integration of cation/solute-binding sites within a common cation/permeant translocation pore, mutation of Glu-343 and Glu-519 also altered hCNT3 Nucleoside transport selectivity. Both residues were accessible to the external medium and inhibited by p-chloromercuribenzene sulfonate when converted to cysteine.
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Conserved Glutamate Residues Glu-343 and Glu-519 Provide Mechanistic Insights into Cation/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter hCNT3
The Journal of biological chemistry, 2009Co-Authors: Melissa D Slugoski, Carol E Cass, Stephen A. Baldwin, Kyla M Smith, Sylvia Y. M. Yao, Edward Karpinski, James D YoungAbstract:Human Concentrative Nucleoside Transporter 3 (hCNT3) utilizes electrochemical gradients of both Na+ and H+ to accumulate pyrimidine and purine Nucleosides within cells. We have employed radioisotope flux and electrophysiological techniques in combination with site-directed mutagenesis and heterologous expression in Xenopus oocytes to identify two conserved pore-lining glutamate residues (Glu-343 and Glu-519) with essential roles in hCNT3 Na+/Nucleoside and H+/Nucleoside cotransport. Mutation of Glu-343 and Glu-519 to aspartate, glutamine, and cysteine severely compromised hCNT3 transport function, and changes included altered Nucleoside and cation activation kinetics (all mutants), loss or impairment of H+ dependence (all mutants), shift in Na+:Nucleoside stoichiometry from 2:1 to 1:1 (E519C), complete loss of catalytic activity (E519Q) and, similar to the corresponding mutant in Na+-specific hCNT1, uncoupled Na+ currents (E343Q). Consistent with close-proximity integration of cation/solute-binding sites within a common cation/permeant translocation pore, mutation of Glu-343 and Glu-519 also altered hCNT3 Nucleoside transport selectivity. Both residues were accessible to the external medium and inhibited by p-chloromercuribenzene sulfonate when converted to cysteine.
Carol E Cass - One of the best experts on this subject based on the ideXlab platform.
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substituted cysteine accessibility method scam analysis of the transport domain of human Concentrative Nucleoside Transporter 3 hcnt3 and other family members reveals features of structural and functional importance
Journal of Biological Chemistry, 2017Co-Authors: Ras Mulinta, Carol E Cass, Amy M L Ng, James D YoungAbstract:Abstract The human SLC28 family of Concentrative Nucleoside Transporter (CNT) proteins has three members: hCNT1, hCNT2, and hCNT3. Na+-coupled hCNT1 and hCNT2 transport pyrimidine and purine Nucleosides, respectively, whereas hCNT3 transports both pyrimidine and purine Nucleosides utilizing Na+ and/or H+ electrochemical gradients. Escherichia coli CNT family member NupC resembles hCNT1 in permeant selectivity but is H+-coupled. Using heterologous expression in Xenopus oocytes and the engineered cysteine-less hCNT3 protein hCNT3(C−), substituted cysteine accessibility method analysis with the membrane-impermeant thiol reactive reagent p-chloromercuribenzene sulfonate was performed on the transport domain (interfacial helix 2, hairpin 1, putative transmembrane domain (TM) 7, and TM8), as well as TM9 of the scaffold domain of the protein. This systematic scan of the entire C-terminal half of hCNT3(C−) together with parallel studies of the transport domain of wild-type hCNT1 and the corresponding TMs of cysteine-less NupC(C−) yielded results that validate the newly developed structural homology model of CNT membrane architecture for human CNTs, revealed extended conformationally mobile regions within transport-domain TMs, identified pore-lining residues of functional importance, and provided evidence of an emerging novel elevator-type mechanism of Transporter function.
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Human Concentrative Nucleoside Transporter 3 Transfection with Ultrasound and Microbubbles in Nucleoside Transport Deficient HEK293 Cells Greatly
2016Co-Authors: Increases Gemcitabine Uptake, James D Young, Carol E Cass, Sylvia Y. M. Yao, Robert J. Paproski, Nicole Favis, David Evans, Roger J. ZempAbstract:Gemcitabine is a hydrophilic clinical anticancer drug that requires Nucleoside Transporters to cross plasma membranes and enter cells. Pancreatic adenocarcinomas with low levels of Nucleoside Transporters are generally resistant to gemcitabine and are currently a clinical problem. We tested whether transfection of human Concentrative Nucleoside Transporter 3 (hCNT3) using ultrasound and lipid stabilized microbubbles could increase gemcitabine uptake and sensitivity in HEK293 cells made Nucleoside transport deficient by pharmacologic treatment with dilazep. To our knowledge, no published data exists regarding the utility of using hCNT3 as a therapeutic gene to reverse gemcitabine resistance. Our ultrasound transfection system- capable of transfection of cell cultures, mouse muscle and xenograft CEM/araC tumors- increased hCNT3 mRNA and 3H-gemcitabine uptake by.2,000 – and 3,400–fold, respectively, in dilazep-treated HEK293 cells. Interestingly, HEK293 cells with both functional human equilibrative Nucleoside Transporters and hCNT3 displayed 5 % of 3H-gemcitabine uptake observed in cells with only functional hCNT3, suggesting that equilibrative Nucleoside Transporters caused significant efflux of 3H-gemcitabine. Efflux assays confirmed that dilazep could inhibit the majority of 3H-gemcitabine efflux from HEK293 cells, suggesting that hENTs were responsible for the majority of efflux from the tested cells. Oocyte uptake transport assays were also performed and provided support for our hypothesis. Gemcitabine uptake and efflux assays were also performed on pancreatic cancer AsPC-1 and MIA PaCa-2 cells with similar results to that o
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human Concentrative Nucleoside Transporter 3 transfection with ultrasound and microbubbles in Nucleoside transport deficient hek293 cells greatly increases gemcitabine uptake
PLOS ONE, 2013Co-Authors: Robert J. Paproski, James D Young, Carol E Cass, Nicole Favis, David H Evans, Roger J. ZempAbstract:Gemcitabine is a hydrophilic clinical anticancer drug that requires Nucleoside Transporters to cross plasma membranes and enter cells. Pancreatic adenocarcinomas with low levels of Nucleoside Transporters are generally resistant to gemcitabine and are currently a clinical problem. We tested whether transfection of human Concentrative Nucleoside Transporter 3 (hCNT3) using ultrasound and lipid stabilized microbubbles could increase gemcitabine uptake and sensitivity in HEK293 cells made Nucleoside transport deficient by pharmacologic treatment with dilazep. To our knowledge, no published data exists regarding the utility of using hCNT3 as a therapeutic gene to reverse gemcitabine resistance. Our ultrasound transfection system - capable of transfection of cell cultures, mouse muscle and xenograft CEM/araC tumors - increased hCNT3 mRNA and 3H-gemcitabine uptake by >2,000– and 3,400–fold, respectively, in dilazep-treated HEK293 cells. Interestingly, HEK293 cells with both functional human equilibrative Nucleoside Transporters and hCNT3 displayed 5% of 3H-gemcitabine uptake observed in cells with only functional hCNT3, suggesting that equilibrative Nucleoside Transporters caused significant efflux of 3H-gemcitabine. Efflux assays confirmed that dilazep could inhibit the majority of 3H-gemcitabine efflux from HEK293 cells, suggesting that hENTs were responsible for the majority of efflux from the tested cells. Oocyte uptake transport assays were also performed and provided support for our hypothesis. Gemcitabine uptake and efflux assays were also performed on pancreatic cancer AsPC-1 and MIA PaCa-2 cells with similar results to that of HEK293 cells. Using the MTS proliferation assay, dilazep-treated HEK293 cells demonstrated 13-fold greater resistance to gemcitabine compared to dilazep-untreated HEK293 cells and this resistance could be reversed by transfection of hCNT3 cDNA. We propose that transfection of hCNT3 cDNA using ultrasound and microbubbles may be a method to reverse gemcitabine resistance in pancreatic tumors that have little Nucleoside transport activity which are resistant to almost all current anticancer therapies.
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conserved glutamate residues glu 343 and glu 519 provide mechanistic insights into cation Nucleoside cotransport by human Concentrative Nucleoside Transporter hcnt3
Journal of Biological Chemistry, 2009Co-Authors: Melissa D Slugoski, Carol E Cass, Stephen A. Baldwin, Kyla M Smith, Sylvia Y. M. Yao, Edward Karpinski, James D YoungAbstract:Human Concentrative Nucleoside Transporter 3 (hCNT3) utilizes electrochemical gradients of both Na+ and H+ to accumulate pyrimidine and purine Nucleosides within cells. We have employed radioisotope flux and electrophysiological techniques in combination with site-directed mutagenesis and heterologous expression in Xenopus oocytes to identify two conserved pore-lining glutamate residues (Glu-343 and Glu-519) with essential roles in hCNT3 Na+/Nucleoside and H+/Nucleoside cotransport. Mutation of Glu-343 and Glu-519 to aspartate, glutamine, and cysteine severely compromised hCNT3 transport function, and changes included altered Nucleoside and cation activation kinetics (all mutants), loss or impairment of H+ dependence (all mutants), shift in Na+:Nucleoside stoichiometry from 2:1 to 1:1 (E519C), complete loss of catalytic activity (E519Q) and, similar to the corresponding mutant in Na+-specific hCNT1, uncoupled Na+ currents (E343Q). Consistent with close-proximity integration of cation/solute-binding sites within a common cation/permeant translocation pore, mutation of Glu-343 and Glu-519 also altered hCNT3 Nucleoside transport selectivity. Both residues were accessible to the external medium and inhibited by p-chloromercuribenzene sulfonate when converted to cysteine.
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Conserved Glutamate Residues Glu-343 and Glu-519 Provide Mechanistic Insights into Cation/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter hCNT3
The Journal of biological chemistry, 2009Co-Authors: Melissa D Slugoski, Carol E Cass, Stephen A. Baldwin, Kyla M Smith, Sylvia Y. M. Yao, Edward Karpinski, James D YoungAbstract:Human Concentrative Nucleoside Transporter 3 (hCNT3) utilizes electrochemical gradients of both Na+ and H+ to accumulate pyrimidine and purine Nucleosides within cells. We have employed radioisotope flux and electrophysiological techniques in combination with site-directed mutagenesis and heterologous expression in Xenopus oocytes to identify two conserved pore-lining glutamate residues (Glu-343 and Glu-519) with essential roles in hCNT3 Na+/Nucleoside and H+/Nucleoside cotransport. Mutation of Glu-343 and Glu-519 to aspartate, glutamine, and cysteine severely compromised hCNT3 transport function, and changes included altered Nucleoside and cation activation kinetics (all mutants), loss or impairment of H+ dependence (all mutants), shift in Na+:Nucleoside stoichiometry from 2:1 to 1:1 (E519C), complete loss of catalytic activity (E519Q) and, similar to the corresponding mutant in Na+-specific hCNT1, uncoupled Na+ currents (E343Q). Consistent with close-proximity integration of cation/solute-binding sites within a common cation/permeant translocation pore, mutation of Glu-343 and Glu-519 also altered hCNT3 Nucleoside transport selectivity. Both residues were accessible to the external medium and inhibited by p-chloromercuribenzene sulfonate when converted to cysteine.
Marcal Pastoranglada - One of the best experts on this subject based on the ideXlab platform.
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link between high affinity adenosine Concentrative Nucleoside Transporter 2 cnt2 and energy metabolism in intestinal and liver parenchymal cells
Journal of Cellular Physiology, 2010Co-Authors: Isabel Huberruano, Javier F Casado, Gonzalo E. Torres, Itziar Pinillamacua, Marcal PastorangladaAbstract:Concentrative Nucleoside Transporter 2 (CNT2) is a high-affinity adenosine Transporter that may play physiological roles beyond Nucleoside salvage. Previous reports relate CNT2 function to modulation of purinergic signaling and energy metabolism in intestinal and liver parenchymal cells (Duflot et al., 2004, Mol Cell Biol 24:2710-2719; Aymerich et al., 2006, J Cell Sci 119:1612-1621). In the present study, to further examine the link between CNT2 and energy metabolism, CNT2 protein partners were identified using the bacterial two-hybrid and GST pull-down approaches. The N-terminal segment of CNT2 was used as bait, since proteins lacking this domain display impaired plasma membrane insertion and intracellular retention. Glucose-regulated protein 58 (GRP58) was identified as a potential rCNT2 partner in pull-down experiments. Two-hybrid screening performed against a liver human cDNA library led to the identification of aldolase B as another hCNT2 partner. Aldolase B-RFP and endogenous GRP58 separately co-localized with CNT2 in HeLa cells transfected with YFPrCNT2. CNT2 interaction with GRP58 was validated using co-immunoprecipitation experiments. In HeLa cells, fluorescence resonance energy transfer (FRET) efficiency increased upon fructose addition, consistent with a transient interaction between aldolase B and the Transporter. The physiological basis for in vivo interactions was derived from experiments in which GRP58 was inhibited or overexpressed and aldolase B activity stimulated towards glycolysis. GRP58 appeared to be a negative effector of CNT2 function, whereas aldolase B flux modulated CNT2 activity via a mechanism involving acquisition of higher affinity for its substrates. These findings support the theory that CNT2 plays roles other than salvage and establishes links with energy metabolism.
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effects of na and h on steady state and presteady state currents of the human Concentrative Nucleoside Transporter 3 hcnt3
Pflügers Archiv: European Journal of Physiology, 2010Co-Authors: Edurne Gorraitz, Marcal Pastoranglada, M P LostaoAbstract:Human Concentrative Nucleoside Transporter 3 (hCNT3) uses the electrochemical gradient of Na+ and H+ to drive the transport of Nucleosides and therapeutic Nucleoside analogs into the cells. We employed the two-electrode voltage clamp technique to compare the steady-state and presteady-state kinetics of hCNT3 in the presence of Na+ and H+. We found that H+ supported a higher maximal rate of uridine transport than Na+, but the efficiency of transport was lower. For both cations, maximal uridine-induced current increased with hyperpolarizing potentials and did not saturate within the voltage range tested. Apparent affinity of hCNT3 for uridine in H+ was insensitive to membrane voltage at negative potentials, and decreased with depolarization. In contrast, apparent affinity for uridine in Na+ decreased with hyperpolarization and was independent of voltage at depolarizing potentials. H+-coupled hCNT3 exhibited lower affinity for all natural Nucleosides and different substrate selectivity compared to Na+-coupled hCNT3. In H+, lack of the hydroxyl groups at 2′ and 5′ decreased the affinity, while lack of the nitrogen N-7 or inversion of the configuration of the hydroxyl group at 2′ prevented transport. Presteady-state charge movements of hCNT3 did not decrease when extracellular cation concentration (Na+ or H+) was reduced, but the τ ON–V and Q–V curves were shifted to more negative potentials. The different effects of uridine and inosine on presteady-state currents in H+ indicated a change in rate-limiting step for the transport of these substrates by H+-coupled hCNT3.
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all trans retinoic acid promotes trafficking of human Concentrative Nucleoside Transporter 3 hcnt3 to the plasma membrane by a tgf β1 mediated mechanism
Journal of Biological Chemistry, 2010Co-Authors: Paula Fernandezcalotti, Marcal PastorangladaAbstract:Human Concentrative Nucleoside Transporter-3 (hCNT3) is a sodium-coupled Nucleoside Transporter that exhibits high affinity and broad substrate selectivity, making it the most suitable candidate for mediating the uptake and cytotoxic action of most Nucleoside-derived drugs. The drug of this class most commonly used in the treatment of chronic lymphocytic leukemia (CLL) is the pro-apoptotic Nucleoside analog fludarabine (Flu), which enters CLL cells primarily through human equilibrative Nucleoside Transporters (hENTs). Although CLL cells lack hCNT3 activity, they do express this Transporter protein, which is located mostly in the cytosol. The aim of our study was to identify agents and mechanisms capable of promoting hCNT3 trafficking to the plasma membrane. Here, we report that all-trans-retinoic acid (ATRA), currently used in the treatment of acute promyelocytic leukemia (APL), increases hCNT3-related activity through a mechanism that involves trafficking of pre-existing hCNT3 proteins to the plasma membrane. This effect is mediated by the autocrine action of transforming growth factor (TGF)-β1, which is transcriptionally activated by ATRA in a p38-dependent manner. TGF-β1 acts through activation of ERK1/2 and the small GTPase RhoA to promote plasma membrane trafficking of the hCNT3 protein.
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role of the human Concentrative Nucleoside Transporter hcnt1 in the cytotoxic action of 5 prime deoxy 5 fluorouridine an active intermediate metabolite of capecitabine a novel oral anticancer drug
Molecular Pharmacology, 2001Co-Authors: Joao F Mata, Jose Manuel Garciamanteiga, Pilar M Lostao, Sonia Fernandezveledo, Elena Guillengomez, Ignacio M Larrayoz, Jorge Lloberas, Javier F Casado, Marcal PastorangladaAbstract:We attempt to identify the plasma membrane Transporter involved in the uptake of 5′-deoxy-5-fluorouridine (5′-DFUR), an intermediate metabolite of capecitabine. This novel oral fluoropyrimidine is used in cancer treatments and is a direct precursor of the cytostatic agent 5′-fluorouracil. We also examine the role of the Transporter in 5′-DFUR cytotoxicity. The human Concentrative Nucleoside Transporter (hCNT1) was cloned from human fetal liver and expressed in Xenopus laevis oocytes. The two-electrode voltage-clamp technique was used to demonstrate that 5′-DFUR, but not capecitabine or 5′-FU, is an hCNT1 substrate. Then, hCNT1 was heterologously expressed in the mammalian cell line Chinese hamster ovary-K1. Functional expression was demonstrated by monitoring transport of radiolabeled substrates and by using a monospecific polyclonal antibody generated against the Transporter. hCNT1-expressing cells were more sensitive to 5′-DFUR than vector-transfected or wild-type cells. The sensitivity of the three cell types to other agents such as cisplatin or 5′-FU was identical. In conclusion, this study shows that 1) the pharmacological profile of a Nucleoside Transporter can be determined by an electrophysiological approach; 2) the hCNT1 Transporter is involved in 5′-DFUR uptake; and 3) hCNT1 expression may increase cell sensitivity to 5′-DFUR treatment. This study also reports for the first time the generation of an antibody against hCNT1, which may be useful in the elucidation of the relationship between hCNT1 expression and tumor response to capecitabine treatment.
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hormonal regulation of Concentrative Nucleoside transport in liver parenchymal cells
Biochemical Journal, 1996Co-Authors: M Gomezangelats, B Del Santo, J Mercader, Andreu Ferrermartinez, Antonio Felipe, J Casado, Marcal PastorangladaAbstract:: Na(+)-dependent uridine uptake is stimulated in isolated rat liver parenchymal cells by glucagon. This effect is transient, reaches maximum levels of stimulation 10 min after hormone addition, and is dose-dependent. Glucagon action can be mimicked by agents that are able to hyperpolarize the plasma membrane (e.g. monensin) and by dibutyryl cyclic AMP. The effects triggered by glucagon, monensin and dibutyryl cyclic AMP are not additive, suggesting a common mechanism of action. 8-(4-Chloro-phenylthio)adenosine 3':5'-cyclic monophosphate (PCT), a cyclic AMP analogue but also a Nucleoside analogue, markedly stimulates Na(+)-dependent uridine uptake in an additive manner to that triggered by monensin, similarly to the effect described for nitrobenzylthioinosine. Considering the roles reported for Nucleosides in liver metabolism, the use of PCT as a cyclic AMP analogue should be precluded. Insulin is also about to up-regulate Na(+)-dependent uridine uptake by a mechanism which involves a stable induction of this transport activity at the plasma-membrane level. This is consistent with a mechanism involving synthesis and insertion of more carriers into the plasma membrane. It is concluded that the recently characterized hepatic Concentrative Nucleoside Transporter is under short-term hormonal regulation by glucagon, through mechanisms which involve membrane hyperpolarization, and under long-term control by insulin. This is the first report showing hormonal modulation of the hepatic Concentrative Nucleoside Transporter.
Kathleen M. Giacomini - One of the best experts on this subject based on the ideXlab platform.
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Human Concentrative Nucleoside Transporter 3 (hCNT3, SLC28A3) Forms a Cyclic Homotrimer
Biochemistry, 2017Co-Authors: Adrian Stecula, Avner Schlessinger, Kathleen M. Giacomini, Andrej SaliAbstract:Many anticancer and antiviral drugs are purine or pyrimidine analogues, which use membrane Transporters to cross cellular membranes. Concentrative Nucleoside Transporters (CNTs) mediate the salvage of Nucleosides and the transport of therapeutic Nucleoside analogues across plasma membranes by coupling the transport of ligands to the sodium gradient. Of the three members of the human CNT family, CNT3 has the broadest selectivity and the widest expression profile. However, the molecular mechanisms of the Transporter, including how it interacts with and translocates structurally diverse Nucleosides and Nucleoside analogues, are unclear. Recently, the crystal structure of vcCNT showed that the prokaryotic homologue of CNT3 forms a homotrimer. In this study, we successfully expressed and purified the wild type human homologue, hCNT3, demonstrating the homotrimer by size exclusion profiles and glutaraldehyde cross-linking. Further, by creating a series of cysteine mutants at highly conserved positions guided by comparative structure models, we cross-linked hCNT3 protomers in a cell-based assay, thus showing the existence of hCNT3 homotrimers in human cells. The presence and absence of cross-links at specific locations along TM9 informs us of important structural differences between vcCNT and hCNT3. Comparative modeling of the trimerization domain and sequence coevolution analysis both indicate that oligomerization is critical to the stability and function of hCNT3. In particular, trimerization appears to shorten the translocation path for Nucleosides across the plasma membrane and may allow modulation of the transport function via allostery.
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Molecular determinants of specificity for synthetic Nucleoside analogs in the Concentrative Nucleoside Transporter, CNT2
The Journal of biological chemistry, 2006Co-Authors: Ryan P. Owen, Ilaria Badagnani, Kathleen M. GiacominiAbstract:Members of the Concentrative Nucleoside Transporter (CNT) family (SLC28) mediate the transport of naturally-occurring Nucleosides, and Nucleoside analog drugs across the plasma membrane of epithelial cells. Each of the three CNT family members has a distinct specificity for naturally occurring Nucleosides, and residues that contribute to the specificity of each Transporter have been identified. In contrast, the molecular determinants of specificity for synthetic Nucleoside analogs are not known. In this study, we take advantage of the large species difference that exists between human and rat CNT2 (hCNT2 and rCNT2) in their ability to transport the Nucleoside analog drug cladribine, 2CdA, (rCNT2 > > > hCNT2) to identify the critical domains and amino acid residues that contribute to the observed difference in specificity between CNT2 orthologs. Using chimeric proteins of human and rat CNT2, we determined that the C-terminal half of CNT2 contained the determinants of 2CdA selectivity. We replaced key residues in the C terminus of hCNT2 with the equivalent residue in rCNT2. One residue in the C-terminal portion of CNT2 was found to significantly contribute to 2CdA selectivity: hCNT2-S354A. This mutant caused an increase of 5-6-fold over hCNT2. The 2-chloro pharmacophore, rather than the 2'-deoxyribose was responsible for the reduced 2CdA uptake by hCNT2. Our data are consistent with a model in which an increased capability for hydrogen bonding in critical amino acids that reside in the C terminus of rCNT2 contributes to its enhanced selectivity for 2CdA.
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Genetic analysis and functional characterization of polymorphisms in the human Concentrative Nucleoside Transporter, CNT2.
Pharmacogenetics and genomics, 2005Co-Authors: Ryan P. Owen, C C Huang, D Stryke, M Kawamoto, S J Johns, T E Ferrin, E J Carlson, Jennifer H. Gray, Travis R. Taylor, Kathleen M. GiacominiAbstract:The Concentrative Nucleoside Transporter CNT2 (SPNT1; SLC28A2) plays a role in the absorption and disposition of naturally occurring Nucleosides, as well as Nucleoside analog drugs. The aim of the present study was to characterize genetic variation in SLC28A2, the gene encoding CNT2, and to functionally analyse non-synonymous variants of CNT2, as a first step towards understanding whether genetic variation in this Nucleoside Transporter contributes to variation in response to Nucleoside analogs. As part of a larger study, DNA samples from an ethnically diverse population (100 African-Americans, 100 European-Americans, 30 Asians, 10 Mexicans and seven Pacific Islanders) were screened and 10 coding region variants of CNT2 were identified. The non-synonymous variants were then constructed and characterized in Xenopus laevis oocytes. Six non-synonymous variants were identified, and all were able to transport guanosine. The four common variants (>1% in the sample population) were further characterized with the anti-viral Nucleoside analog drug ribavirin. No differences were observed among the four common variants in the uptake kinetics of 3H-ribavirin (Km in microM: 35.6+/-9.27 for CNT2-reference, 40.7+/-6.47 for CNT2-P22L, 31.2+/-15.8 for CNT2-S75R, 26.7+/-6.13 for CNT2-S245T and 49.9+/-14.6 for CNT2-F355S). The variant CNT2-F355S exhibited a change in specificity for the naturally occurring Nucleosides, inosine and uridine. All non-synonymous variants of CNT2 took up guanosine, and the four variants examined showed no significant difference in ribavirin kinetics. However, CNT2-F355S (3% allele frequency in the African-American sample) was found to alter specificity for naturally occurring Nucleosides, which may have implications for Nucleoside homeostasis.
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The Concentrative Nucleoside Transporter family, SLC28
Pflügers Archiv, 2004Co-Authors: Jennifer H. Gray, Ryan P. Owen, Kathleen M. GiacominiAbstract:The SLC28 family consists of three subtypes of sodium-dependent, Concentrative Nucleoside Transporters, CNT1, CNT2, and CNT3 (SLC28A1, SLC28A2, and SLC28A3, respectively), that transport both naturally occurring Nucleosides and synthetic Nucleoside analogs used in the treatment of various diseases. These subtypes differ in their substrate specificities: CNT1 is pyrimidine-Nucleoside preferring, CNT2 is purine-Nucleoside preferring, and CNT3 transports both pyrimidine and purine Nucleosides. Recent studies have identified key amino acid residues that are determinants of pyrimidine and purine specificity of CNT1 and CNT2. The tissue distributions of the CNTs vary: CNT1 is localized primarily in epithelia, whereas CNT2 and CNT3 have more generalized distributions. Nucleoside Transporters in the SLC28 and SLC29 families play critical roles in Nucleoside salvage pathways where they mediate the first step of nucleotide biosynthesis. In addition, these Transporters work in concert to terminate adenosine signaling. SLC28 family members are crucial determinants of response to a variety of anticancer and antiviral Nucleoside analogs, as they modulate the entry of these analogs into target tissues. Further, this family is involved in the absorption and disposition of many Nucleoside analogs. Several CNT single Nucleoside polymorphisms (SNPs) have been identified, but have yet to be characterized.
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Interaction of cladribine and fludarabine with genetic variants in the Concentrative Nucleoside Transporter, CNT3
Clinical Pharmacology & Therapeutics, 2004Co-Authors: I Badagnani, Kathleen M. GiacominiAbstract:The Concentrative Nucleoside Transporter, CNT3 (SLC28A3), mediates the intracellular uptake of the anti-cancer Nucleoside analogs, cladribine and fludarabine. Wide variation in intracellular levels of these analogs has been described. To test the hypothesis that coding variants in CNT3 may contribute to this phenotype, we identified and functionally tested missense variants of CNT3. Ten missense variants were found in a collection of 256 ethnically diverse DNA samples; three of these had total allele frequencies ≥1%. The activity of the missense variants was determined by isotopic uptake of model Nucleosides in X. laevis oocytes expressing the CNT3 variants. The rare variant, CNT3-G>R, showed an 85% and 83% reduction in the transport of inosine and thymidine, respectively. All other variants had similar activity as the reference. There was no difference in the interaction kinetics of the three common variants with adenosine when compared to reference (Km=69+11, 91+24, 78+9, 94+3 μM for CNT3, CNT3-S>N, -Y>C and -I>V, respectively). These studies indicate that common missense variants of CNT3 have similar activities as the reference and suggest that they do not contribute to variation in intracellular levels of Nucleosides and Nucleoside analogs. Studies are underway to assess the interaction kinetics of Nucleoside analogs with the common CNT3 variants and to test whether variation in CNT3 expression contributes to variation in intracellular levels of Nucleoside analogs. Clinical Pharmacology & Therapeutics (2004) 75, P18–P18; doi: 10.1016/j.clpt.2003.11.066