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Hiroaki Yuasa - One of the best experts on this subject based on the ideXlab platform.
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Current Understanding of the Intestinal Absorption of Nucleobases and Analogs.
Biological & pharmaceutical bulletin, 2020Co-Authors: Hiroaki Yuasa, Tomoya Yasujima, Katsuhisa InoueAbstract:It has long been suggested that a Na+-dependent carrier-mediated transport system is involved in the absorption of Nucleobases and analogs, including some drugs currently in therapeutic use, for their uptake at the brush border membrane of epithelial cells in the small intestine, mainly based on studies in non-primate experimental animals. The presence of this transport system was indeed proved by the recent identification of sodium-dependent Nucleobase Transporter 1 (SNBT1/Slc23a4) as its molecular entity in rats. However, this Transporter has been found to be genetically deficient in humans and higher primates. Aware of this deficiency, we need to revisit the issue of the absorption of these compounds in the human small intestine so that we can understand the mechanisms and gain information to assure the more rational use and development of drugs analogous to Nucleobases. Here, we review the current understanding of the intestinal absorption of Nucleobases and analogs. This includes recent knowledge about the efflux transport of those compounds across the basolateral membrane when exiting epithelial cells, following brush border uptake, in order to complete the overall absorption process; the facilitative Transporters of equilibrative nucleoside Transporter 1 (ENT1/SLC29A1) and equilibrative Nucleobase Transporter 1 (ENBT1/SLC43A3) may be involved in that in many animal species, including human and rat, without any major species differences.
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functional analysis of the role of equilibrative Nucleobase Transporter 1 enbt1 slc43a3 in adenine transport in hepg2 cells
Journal of Pharmaceutical Sciences, 2020Co-Authors: Risa Takenaka, Katsuhisa Inoue, Tomoya Yasujima, Kinya Ohta, Junji Furukawa, Yosuke Hishikawa, Takahiro Yamashiro, Hiroaki YuasaAbstract:Abstract Equilibrative Nucleobase Transporter 1 (ENBT1/SLC43A3) has recently been identified as a purine-selective Nucleobase Transporter. Although it is highly expressed in the liver, its role in Nucleobase transport has not been confirmed yet in hepatocytes or any relevant cell models. We, therefore, examined its role in adenine transport in the HepG2 cell line as a human hepatocyte model. The uptake of [3H]adenine in HepG2 cells was highly saturable, indicating the involvement of carrier-mediated transport. The carrier-mediated transport component, for which the Michaelis constant was estimated to be 0.268 μM, was sensitive to decynium-22, an ENBT1 inhibitor, with the half maximal inhibitory concentration of 2.59 μM, which was comparable to that of 2.30 μM for [3H]adenine uptake by ENBT1 in its transient transfectant human embryonic kidney 293 cells. Although equilibrative nucleoside Transporter 1 (ENT1/SLC29A1) and ENT2/SLC29A2 are also known to be able to transport adenine, [3H]adenine uptake in HepG2 cells was not inhibited by the ENT1/2-specific inhibitor of either dipyridamole or nitrobenzylthioinosine. Finally, [3H]adenine uptake was extensively reduced by silencing of ENBT1 by RNA interference in the hepatocyte model. All these results, taken together, suggest the predominant role of ENBT1 in the uptake of adenine in HepG2 cells.
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Functional Analysis of the Role of Equilibrative Nucleobase Transporter 1 (ENBT1/SLC43A3) in Adenine Transport in HepG2 Cells
Journal of pharmaceutical sciences, 2020Co-Authors: Risa Takenaka, Katsuhisa Inoue, Tomoya Yasujima, Kinya Ohta, Junji Furukawa, Yosuke Hishikawa, Takahiro Yamashiro, Hiroaki YuasaAbstract:Abstract Equilibrative Nucleobase Transporter 1 (ENBT1/SLC43A3) has recently been identified as a purine-selective Nucleobase Transporter. Although it is highly expressed in the liver, its role in Nucleobase transport has not been confirmed yet in hepatocytes or any relevant cell models. We, therefore, examined its role in adenine transport in the HepG2 cell line as a human hepatocyte model. The uptake of [3H]adenine in HepG2 cells was highly saturable, indicating the involvement of carrier-mediated transport. The carrier-mediated transport component, for which the Michaelis constant was estimated to be 0.268 μM, was sensitive to decynium-22, an ENBT1 inhibitor, with the half maximal inhibitory concentration of 2.59 μM, which was comparable to that of 2.30 μM for [3H]adenine uptake by ENBT1 in its transient transfectant human embryonic kidney 293 cells. Although equilibrative nucleoside Transporter 1 (ENT1/SLC29A1) and ENT2/SLC29A2 are also known to be able to transport adenine, [3H]adenine uptake in HepG2 cells was not inhibited by the ENT1/2-specific inhibitor of either dipyridamole or nitrobenzylthioinosine. Finally, [3H]adenine uptake was extensively reduced by silencing of ENBT1 by RNA interference in the hepatocyte model. All these results, taken together, suggest the predominant role of ENBT1 in the uptake of adenine in HepG2 cells.
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Urate transport function of rat sodium-dependent Nucleobase Transporter 1.
Physiological reports, 2018Co-Authors: Tomoya Yasujima, Katsuhisa Inoue, Chihiro Murata, Yoshihisa Mimura, Tomoaki Murata, Masahiko Ohkubo, Kinya Ohta, Hiroaki YuasaAbstract:Sodium-dependent Nucleobase Transporter 1 (SNBT1) is a Nucleobase-specific Transporter identified in our recent study. In an attempt to search for its potential substrates other than Nucleobases in this study, we could successfully find urate, a metabolic derivative of purine Nucleobases, as a novel substrate, as indicated by its specific Na+ -dependent and saturable transport, with a Michaelis constant of 433 μmol/L, by rat SNBT1 (rSNBT1) stably expressed in Madin-Darby canine kidney II cells. However, urate uptake was observed only barely in the everted tissue sacs of the rat small intestine, in which rSNBT1 operates for Nucleobase uptake. These findings suggested that urate undergoes a futile cycle, in which urate transported into epithelial cells is immediately effluxed back by urate efflux Transporters, in the small intestine. In subsequent attempts to examine that possibility, such a futile urate cycle was demonstrated in the human embryonic kidney 293 cell line as a model cell system, where urate uptake induced by transiently introduced rSNBT1 was extensively reduced by the co-introduction of rat breast cancer resistance protein (rBCRP), a urate efflux Transporter present in the small intestine. However, urate uptake was not raised in the presence of Ko143, a BCRP inhibitor, in the everted intestinal tissue sacs, suggesting that some other Transporter might also be involved in urate efflux. The newly found urate transport function of SNBT1, together with the suggested futile urate cycle in the small intestine, should be of interest for its evolutional and biological implications, although SNBT1 is genetically deficient in humans.
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Dataset for: Urate transport function of rat sodium-dependent Nucleobase Transporter 1
2018Co-Authors: Tomoya Yasujima, Katsuhisa Inoue, Chihiro Murata, Yoshihisa Mimura, Tomoaki Murata, Masahiko Ohkubo, Kinya Ohta, Hiroaki YuasaAbstract:Sodium-dependent Nucleobase Transporter 1 (SNBT1) is a Nucleobase-specific Transporter identified in our recent study. In an attempt to search for its potential substrates other than Nucleobases in the present study, we could successfully find urate, a metabolic derivative of purine Nucleobases, as a novel substrate, as indicated by its specific Na+-dependent and saturable transport, with a Michaelis constant of 433 μM, by rat SNBT1 (rSNBT1) stably expressed in Madin-Darby canine kidney II cells. However, urate uptake was observed only barely in the everted tissue sacs of the rat small intestine, in which rSNBT1 operates for Nucleobase uptake. These findings suggested that urate undergoes a futile cycle, in which urate transported into epithelial cells is immediately effluxed back by urate efflux Transporters, in the small intestine. In subsequent attempts to examine that possibility, such a futile urate cycle was demonstrated in the human embryonic kidney 293 cell line as a model cell system, where urate uptake induced by transiently introduced rSNBT1 was extensively reduced by the co-introduction of rat breast cancer resistance protein (rBCRP), a urate efflux Transporter present in the small intestine. However, urate uptake was not raised in the presence of Ko143, a BCRP inhibitor, in the everted intestinal tissue sacs, suggesting that some other Transporter might also be involved in urate efflux. The newly found urate transport function of SNBT1, together with the suggested futile urate cycle in the small intestine, should be of interest for its evolutional and biological implications, although SNBT1 is genetically deficient in humans
Harry P De Koning - One of the best experts on this subject based on the ideXlab platform.
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Cloning and characterisation of the Equilibrative Nucleoside Transporter family of Trypanosoma cruzi: ultra-high affinity and selectivity to survive in the intracellular niche.
Biochimica et biophysica acta. General subjects, 2018Co-Authors: Gustavo Daniel Campagnaro, Janaina De Freitas Nascimento, Richard M. B. M. Girard, Ariel Mariano Silber, Harry P De KoningAbstract:Abstract Background Trypanosoma cruzi, the causative agent of Chagas' disease is unable to synthesise its own purines and relies on salvage from the host. In other protozoa, purine uptake has been shown to be mediated by Equilibrative Nucleoside Transporters (ENTs). Methods To investigate the functionality of T. cruzi-encoded ENT Transporters, its four putative ENT genes (TcrNB1, TcrNB2, TcrNT1 and TcrNT2) were cloned and expressed in genetically adapted Trypanosoma brucei procyclic cells from which the Nucleobase Transporter locus was deleted. Results TcrNB1 displayed very high affinity for hypoxanthine (Km 93.8 ± 4.7 nM for) and guanine, and moderate affinity for adenine. TcrNT1 was found to be a high-affinity guanosine/inosine Transporter (inosine Km is 1.0 ± 0.03 μM; guanosine Ki is 0.92 ± 0.2 μM). TcrNT2 encoded a high-affinity thymidine Transporter (Km = 223.5 ± 7.1 nM) with a clear preference for 2’-deoxypyrimidines. TcrNB2, whose activity could not be determined in our system, could be a low-affinity purine Nucleobase Transporter, given its sequence and predicted structural similarities to Leishmania major NT4. All 4 Transporter genes were highly expressed in the amastigote forms, with much lower expression in the non-dividing stages. Conclusions The data appear to show that, surprisingly, T. cruzi has a preference for oxopurines over aminopurines and efficiently transports 2′-deoxypyrimidines. The T. cruzi ENTs display exceptionally high substrate affinity as an adaptation to their intracellular localisation. General significance This study reports the first cloning of T. cruzi purine and pyrimidine Transporters, including the first gene encoding a pyrimidine-selective protozoan Transporter.
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Uptake of purines in Plasmodium falciparum-infected human erythrocytes is mostly mediated by the human Equilibrative Nucleoside Transporter and the
2013Co-Authors: Human Facilitative Nucleobase Transporter, Lisa C. Ranford-cartwright, Neils B. Quashie, Harry P De KoningAbstract:Background: Plasmodium parasites are unable to synthesize purines de novo and have to salvage them from the host. Due to this limitation in the parasite, purine Transporters have been an area of focus in the search for antimalarial drugs. Although the uptake of purines through the human equilibrative nucleoside Transporter (hENT1), the human facilitative Nucleobase Transporter (hFNT1) and the parasite-induced new permeation pathway (NPP) has been studied, no information appears to exist on the relative contribution of these three Transporters to the uptake of adenosine and hypoxanthine. Using the appropriate Transporter inhibitors, the role of each of these salvage pathways to the overall purine transport in intraerythrocytic Plasmodium falciparum was systematically investigated. Methods: The transport of adenosine, hypoxanthine and adenine into uninfected and P. falciparum-infected human erythrocytes was investigated in the presence or absence of classical inhibitors of the hFNT1, hENT1 and NPP. The effective inhibition of the various Transporters by the classical inhibitors was verified using appropriate known substrates. The ability of high concentration of unlabelled substrates to saturate these Transporters was also studied. Results: Transport of exogenous purine into infected or uninfected erythrocytes occurred primarily throug
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Uptake of purines in Plasmodium falciparum -infected human erythrocytes is mostly mediated by the human Equilibrative Nucleoside Transporter and the human Facilitative Nucleobase Transporter
Malaria journal, 2010Co-Authors: Neils B. Quashie, Lisa C. Ranford-cartwright, Harry P De KoningAbstract:Background Plasmodium parasites are unable to synthesize purines de novo and have to salvage them from the host. Due to this limitation in the parasite, purine Transporters have been an area of focus in the search for anti-malarial drugs. Although the uptake of purines through the human equilibrative nucleoside Transporter (hENT1), the human facilitative Nucleobase Transporter (hFNT1) and the parasite-induced new permeation pathway (NPP) has been studied, no information appears to exist on the relative contribution of these three Transporters to the uptake of adenosine and hypoxanthine. Using the appropriate Transporter inhibitors, the role of each of these salvage pathways to the overall purine transport in intraerythrocytic Plasmodium falciparum was systematically investigated.
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Uptake of purines in Plasmodium falciparum-infected human erythrocytes is mostly mediated by the human Equilibrative Nucleoside Transporter and the human Facilitative Nucleobase Transporter
Malaria Journal, 2010Co-Authors: Neils B. Quashie, Lisa C. Ranford-cartwright, Harry P De KoningAbstract:Background Plasmodium parasites are unable to synthesize purines de novo and have to salvage them from the host. Due to this limitation in the parasite, purine Transporters have been an area of focus in the search for anti-malarial drugs. Although the uptake of purines through the human equilibrative nucleoside Transporter (hENT1), the human facilitative Nucleobase Transporter (hFNT1) and the parasite-induced new permeation pathway (NPP) has been studied, no information appears to exist on the relative contribution of these three Transporters to the uptake of adenosine and hypoxanthine. Using the appropriate Transporter inhibitors, the role of each of these salvage pathways to the overall purine transport in intraerythrocytic Plasmodium falciparum was systematically investigated. Methods The transport of adenosine, hypoxanthine and adenine into uninfected and P. falciparum -infected human erythrocytes was investigated in the presence or absence of classical inhibitors of the hFNT1, hENT1 and NPP. The effective inhibition of the various Transporters by the classical inhibitors was verified using appropriate known substrates. The ability of high concentration of unlabelled substrates to saturate these Transporters was also studied. Results Transport of exogenous purine into infected or uninfected erythrocytes occurred primarily through saturable Transporters rather than through the NPP. Hypoxanthine and adenine appeared to enter erythrocytes mainly through the hFNT1 Nucleobase Transporter whereas adenosine entered predominantly through the hENT1 nucleoside Transporter. The rate of purine uptake was approximately doubled in infected cells compared to uninfected erythrocytes. In addition, it was found that the rate of adenosine uptake was considerably higher than the rate of hypoxanthine uptake in infected human red blood cells (RBC). It was also demonstrated that furosemide inhibited the transport of purine bases through hFNT1. Conclusion Collectively, the data obtained in this study clearly show that the endogenous host erythrocyte Transporters hENT1 and hFNT1, rather than the NPP, are the major route of entry of purine into parasitized RBC. Inhibitors of hENT1 and hFNT1, as well as the NPP, should be considered in the development of anti-malarials targeted to purine transport.
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Pyrimidine Transporters of trypanosomes – a class apart?
Trends in Parasitology, 2007Co-Authors: Harry P De KoningAbstract:One question that Bellofatto ([1xPyrimidine transport activities in trypanosomes. Bellofatto, V. Trends Parasitol. 2007; 23: 187–189Abstract | Full Text | Full Text PDF | PubMed | Scopus (10)See all References][1]; this issue) notes from our work on the Trypanosoma brucei pyrimidine Transporters is whether they might be exclusive to pyrimidines or represent broad-specificity purine and pyrimidine Transporters. I believe that these Transporters might indeed be pyrimidine specific. We have previously shown that the T. brucei Transporter TbU1 is not inhibited by purine Nucleobases or nucleosides [2xA highly selective, high affinity Transporter for uracil in Trypanosoma brucei brucei; evidence for proton-dependent transport. de Koning, H.P. and Jarvis, S.M. Biochem. Cell Biol. 1998; 76: 853–858CrossRef | PubMedSee all References][2] and recently reinvestigated the limits of what TbU1 and the equivalent Leishmania major Transporter, LmU1, accept as a substrate [3xIdentification of the first pyrimidine Nucleobase Transporter in Leishmania: similarities with the Trypanosoma brucei U1 Transporter and antileishmanial activity of uracil analogues. Papageorgiou, I.G. et al. Parasitology. 2005; 130: 275–283CrossRef | PubMed | Scopus (19)See all References][3]. The substrate of these Transporters needs to be planar and is extremely limited by size: the only acceptable substitution being a fluor residue at position five. Even this minimal change involves some loss of affinity and if the substitution is chloride or methyl the affinity is entirely lost. Substitutions at position six are equally restricted and it is evident that purine rings will not fit the binding pocket. Uridine seems to bind these Transporters only with the pyrimidine ring, thus explaining its low affinity and low translocation efficiency [3xIdentification of the first pyrimidine Nucleobase Transporter in Leishmania: similarities with the Trypanosoma brucei U1 Transporter and antileishmanial activity of uracil analogues. Papageorgiou, I.G. et al. Parasitology. 2005; 130: 275–283CrossRef | PubMed | Scopus (19)See all References, 4xTrypanosoma brucei: a survey of pyrimidine transport activities. Gudin, S. et al. Exp. Parasitol. 2006; 114: 118–125CrossRef | PubMed | Scopus (19)See all References].We have not attempted such exhaustive characterization of the TbC1 cytosine and U2 uridine Transporters and have yet to test the effects of purines on these carriers. However, the high affinity (Km = 48 ± 9 nM) of C1 for its substrate is informative. If C1 was a purine Nucleobase Transporter then it is probable that we would have identified it in T. brucei procyclics but none of the purine Transporters of T. brucei that have been characterized to date has a high affinity for pyrimidines. Similarly, no purine Transporter with a high affinity for uridine can be identified in procyclic T. b. brucei[5xHypoxanthine uptake through a purine-selective Nucleobase Transporter in Trypanosoma brucei brucei procyclics is driven by protonmotive force. de Koning, H.P. and Jarvis, S.M. Eur. J. Biochem. 1997; 247: 1102–1110CrossRef | PubMedSee all References, 6xMolecular interactions underlying the unusually high affinity of a novel Trypanosoma brucei nucleoside Transporter. Al-Salabi, M.I. et al. Mol. Pharmacol. 2007; 71: 921–929CrossRef | PubMed | Scopus (23)See all References, 7xCloning, heterologous expression and in situ characterization of the first high affinity Nucleobase Transporter from a protozoan. Burchmore, R. et al. J. Biol. Chem. 2003; 278: 23502–23507CrossRef | PubMed | Scopus (33)See all References], although the TbU2 carrier displays a Km value of 4.1 μM for this nucleoside [4xTrypanosoma brucei: a survey of pyrimidine transport activities. Gudin, S. et al. Exp. Parasitol. 2006; 114: 118–125CrossRef | PubMed | Scopus (19)See all References][4]. However, we cannot exclude the possibility of an adenine–cytosine Transporter because most of our assays used radiolabeled hypoxanthine and adenosine rather than adenine as permeant.We are confident that T. brucei and L. major encode genuine pyrimidine Transporters. It is unlikely that these are encoded by equilibrative nucleoside Transporter (ENT) family genes because these are now almost all assigned to purine Nucleobase or nucleoside Transporter activities [6xMolecular interactions underlying the unusually high affinity of a novel Trypanosoma brucei nucleoside Transporter. Al-Salabi, M.I. et al. Mol. Pharmacol. 2007; 71: 921–929CrossRef | PubMed | Scopus (23)See all References, 8xPurine Transporters of protozoa: from biology to therapy. de Koning, H.P. et al. FEMS Microbiol. Rev. 2005; 29: 987–1020CrossRef | PubMed | Scopus (100)See all References]. Exceptions are TbNT3 (GeneDB Tb927.2.6200) and TbNT4 (GeneDB Tb927.2.6220) but transport activity for pyrimidines (or purines) could not be detected when these were expressed in Xenopus laevis oocytes [9xSix related nucleoside/Nucleobase Transporters from Trypanosoma brucei exhibit distinct biochemical functions. Sanchez, M.A. et al. J. Biol. Chem. 2002; 277: 21499–21504CrossRef | PubMed | Scopus (41)See all References][9].Bellofatto is thus correct to highlight that the cloning of the pyrimidine Transporter genes could lead to the identification of a new Transporter family, which, like the ENT family, might be present in both protozoa and their mammalian hosts. Although some pyrimidine-specific Transporter genes have been identified from eukaryotes, these are all part of the family that includes the fur4 uracil and fui1 uridine Transporters of Saccharomyces cerevisiae and the furD uracil Transporter of Aspergillus nidulans[10xNucleobase Transporters: a review. de Koning, H.P. and Diallinas, G. Mol. Membr. Biol. 2000; 17: 75–95CrossRef | PubMedSee all References][10]. BLAST analysis does not identify any paralogues in any protozoan or metazoan genome databases. The only reports of pyrimidine-specific transport activity in metazoa that I am aware of are of a sodium-dependent uracil–thymine Transporter in rat intestinal-membrane vesicles [11xIntestinal brush border transport mechanism of 5-fluorouracil in rats. Yuasa, H. et al. Biol. Pharm. Bull. 1996; 19: 94–99CrossRef | PubMedSee all References][11] and a sodium-dependent pyrimidine Nucleobase Transporter in rat Sertoli cells [12xCharacterisation of novel Na+-dependent Nucleobase Transporter systems at the blood–testis barrier. Kato, R. et al. Am. J. Physiol. Endocrinol. Metab. 2006; 290: E968–E975CrossRef | PubMed | Scopus (14)See all References][12].
George Diallinas - One of the best experts on this subject based on the ideXlab platform.
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Structure-activity relationships in fungal Nucleobases Transporters as dissected by the inhibitory effects of novel purine analogues
European journal of medicinal chemistry, 2018Co-Authors: Efthymios-spyridon Gavriil, George Diallinas, Spyridon Dimitrakis, Georgia Papadaki, Sophia Balaska, George Lambrinidis, Nikolaos Lougiakis, Panagiotis Marakos, Nicole Pouli, Emmanuel MikrosAbstract:Abstract We have previously rationally designed, synthesized and tested a number of 3-deazapurine analogues, which inhibit the ubiquitous fungal Nucleobase Transporter FcyB, through binding in its major substrate binding site, by specifically interacting with Asn163. Here, in an effort to further understand the molecular details of structure-activity relationships in all three major Nucleobase Transporters of fungi, we extend this study by designing, based on our previous experience, synthesizing and testing further 3-deazapurine analogues. We thus identify seven new compounds with relatively high affinity (19–106 μΜ) for the FcyB binding site. Importantly, four of these compounds can also efficiently inhibit AzgA, a structurally and evolutionary distinct, but functionally similar, purine Transporter. Contrastingly, none of the new compounds tested had any effect on the transport activity of the uric acid-xanthine Transporter UapA, albeit this being a structural homologue of AzgA. Besides the apparent importance for understanding how Nucleobase Transporter specificity is determined at the molecular level, our work might constitute a critical step in the design of novel purine-related antifungals.
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Cryptic purine Transporters in Aspergillus nidulans reveal the role of specific residues in the evolution of specificity in the NCS1 family.
Molecular microbiology, 2016Co-Authors: Georgia Sioupouli, Emmanuel Mikros, George Lambrinidis, Sotiris Amillis, George DiallinasAbstract:Summary NCS1 proteins are H+ or Na+ symporters responsible for the uptake of purines, pyrimidines or related metabolites in bacteria, fungi and some plants. Fungal NCS1 are classified into two evolutionary and structurally distinct subfamilies, known as Fur- and Fcy-like Transporters. These subfamilies have expanded and functionally diversified by gene duplications. The Fur subfamily of the model fungus Aspergillus nidulans includes both major and cryptic Transporters specific for uracil, 5-fluorouracil, allantoin or/and uric acid. Here we functionally analyse all four A. nidulans Fcy Transporters (FcyA, FcyC, FcyD and FcyE) with previously unknown function. Our analysis shows that FcyD is moderate-affinity, low-capacity, highly specific adenine Transporter, whereas FcyE contributes to 8-azaguanine uptake. Mutational analysis of FcyD, supported by homology modelling and substrate docking, shows that two variably conserved residues (Leu356 and Ser359) in transmembrane segment 8 (TMS8) are critical for transport kinetics and specificity differences among Fcy Transporters, while two conserved residues (Phe167 and Ser171) in TMS3 are also important for function. Importantly, mutation S359N converts FcyD to a promiscuous Nucleobase Transporter capable of recognizing adenine, xanthine and several Nucleobase analogues. Our results reveal the importance of specific residues in the functional evolution of NCS1 Transporters.
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Kinetic and mutational analysis of the Trypanosoma brucei NBT1 Nucleobase Transporter expressed in Saccharomyces cerevisiae reveals structural similarities between ENT and MFS Transporters.
International journal for parasitology, 2007Co-Authors: I. Papageorgiou, H. P. De Koning, Ketty Soteriadou, George DiallinasAbstract:Parasitic protozoa are unable to synthesise purines de novo and thus depend on the uptake of nucleosides and Nucleobases across their plasma membrane through specific Transporters. A number of nucleoside and Nucleobase Transporters from Trypanosoma brucei brucei and Leishmania major have recently been characterised and shown to belong to the equilibrative nucleoside Transporter (ENT) family. A number of studies have demonstrated the functional importance of particular transmembrane segments (TMS) in nucleoside-specific ENT proteins. TbNBT1, one of only three bona fide Nucleobase-selective members of the ENT family, has previously been shown to be a high-affinity Transporter for purine Nucleobases and guanosine. In this study, we use the Saccharomyces cerevisiae expression system to build a biochemical model of how TbNBT1 recognises Nucleobases. We next performed random in vitro and site-directed mutagenesis to identify residues critical for TbNBT1 function. The identification of residues likely to contribute to permeant binding, when combined with a structural model of TbNBT1 obtained by homology threading, yield a tentative three-dimensional model of the Transporter binding site that is consistent with the binding model emerging from the biochemical data. The model strongly suggests the involvement of TMS5, TMS7 and TMS8 in TbNBT1 function. This situation is very similar to that concerning Transporters of the major facilitator superfamily (MFS), one of which was used as a template for the threading. This point raises the possibility that ENT and MFS carriers, despite being considered evolutionarily distinct, might in fact share similar topologies and substrate translocations pathways.
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Identification of the first pyrimidine Nucleobase Transporter in Leishmania: similarities with the Trypanosoma brucei U1 Transporter and antileishmanial activity of uracil analogues.
Parasitology, 2005Co-Authors: I. G. Papageorgiou, George Diallinas, Laith Yakob, M. I. Al Salabi, K. P. Soteriadou, H. P. De KoningAbstract:While purine transport has been widely studied in protozoa, almost nothing is known about their capacity to salvage pyrimidines. Here, we report a Leishmania major Transporter with high affinity for uracil (Km=0.32+/-0.07 microM) which we designated LmU1. This Transporter displayed a high degree of specificity, as it had virtually no affinity for cytosine, thymine or purine Nucleobases, nor did it transport pyrimidine nucleosides. Highest affinity was for 5-fluorouracil. The results show that the permeant binding site of LmU1 interacts strongly with the keto groups of uracil, as shown by a low affinity for 2-thio- and 4-thiouracil. LmU1 appears to further bind uracil through a weak hydrogen bond with N(1)H of the pyrimidine ring in addition to a stronger H-bond with N(3)H. Substrate binding and selectivity were strikingly similar to that of the U1 Transporter in the related kinetoplastid Trypanosoma brucei. Uracil analogues likely to be transported by LmU1 were also screened for antileishmanial activity, with 5-fluorouracil displaying strong activity against promastigotes and intracellular amastigotes. Overall, the results show that, like purine Nucleobase transport, pyrimidine Nucleobase transport function is very similar in L. major and T. brucei insect forms.
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Nucleobase Transporters (review).
Molecular membrane biology, 2000Co-Authors: H. P. De Koning, George DiallinasAbstract:Purines and pyrimidines play a key role in nucleic acid and nucleotide metabolism of all cells. In addition, they can be used as nitrogen sources in plants and many microorganisms. Transport of Nucleobases across biological membranes is mediated by specific transmembrane transport proteins. Nucleobase Transporters have been identified genetically and/or physiologically in bacteria, fungi, protozoa, algae, plants and mammals. A limited number of bacterial and fungal Transporter genes have been cloned and analysed in great detail at the molecular level. Very recently, Nucleobase Transporters have been identified in plants. In other systems, with less accessible genetics, such as vertebrates and protozoa, no Nucleobase Transporter genes have been identified, and the Transporters have been characterized and classified by physiological and biochemical approaches instead. In this review, it is shown that Nucleobase Transporters and similar sequences of unknown function present in databases constitute three basic families, which will be designated NAT, PRT and PUP. The first includes members from archea, eubacteria, fungi, plants and metazoa, the second is restricted to prokaryotes and fungi, and the last one is only found in plants. Interestingly, mammalian ascorbate Transporters are homologous to NAT sequences. The function of different Nucleobase Transporters is also described, as is how their expression is regulated and what is currently known about their structure-function relationships. Common features emerging from these studies are expected to prove critical in understanding what governs Nucleobase Transporter specificity and in selecting proper model microbial systems for cloning and studying plant, protozoan and mammalian Nucleobase Transporters of agricultural, pharmacological and medical importance.
Tomoya Yasujima - One of the best experts on this subject based on the ideXlab platform.
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Current Understanding of the Intestinal Absorption of Nucleobases and Analogs.
Biological & pharmaceutical bulletin, 2020Co-Authors: Hiroaki Yuasa, Tomoya Yasujima, Katsuhisa InoueAbstract:It has long been suggested that a Na+-dependent carrier-mediated transport system is involved in the absorption of Nucleobases and analogs, including some drugs currently in therapeutic use, for their uptake at the brush border membrane of epithelial cells in the small intestine, mainly based on studies in non-primate experimental animals. The presence of this transport system was indeed proved by the recent identification of sodium-dependent Nucleobase Transporter 1 (SNBT1/Slc23a4) as its molecular entity in rats. However, this Transporter has been found to be genetically deficient in humans and higher primates. Aware of this deficiency, we need to revisit the issue of the absorption of these compounds in the human small intestine so that we can understand the mechanisms and gain information to assure the more rational use and development of drugs analogous to Nucleobases. Here, we review the current understanding of the intestinal absorption of Nucleobases and analogs. This includes recent knowledge about the efflux transport of those compounds across the basolateral membrane when exiting epithelial cells, following brush border uptake, in order to complete the overall absorption process; the facilitative Transporters of equilibrative nucleoside Transporter 1 (ENT1/SLC29A1) and equilibrative Nucleobase Transporter 1 (ENBT1/SLC43A3) may be involved in that in many animal species, including human and rat, without any major species differences.
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functional analysis of the role of equilibrative Nucleobase Transporter 1 enbt1 slc43a3 in adenine transport in hepg2 cells
Journal of Pharmaceutical Sciences, 2020Co-Authors: Risa Takenaka, Katsuhisa Inoue, Tomoya Yasujima, Kinya Ohta, Junji Furukawa, Yosuke Hishikawa, Takahiro Yamashiro, Hiroaki YuasaAbstract:Abstract Equilibrative Nucleobase Transporter 1 (ENBT1/SLC43A3) has recently been identified as a purine-selective Nucleobase Transporter. Although it is highly expressed in the liver, its role in Nucleobase transport has not been confirmed yet in hepatocytes or any relevant cell models. We, therefore, examined its role in adenine transport in the HepG2 cell line as a human hepatocyte model. The uptake of [3H]adenine in HepG2 cells was highly saturable, indicating the involvement of carrier-mediated transport. The carrier-mediated transport component, for which the Michaelis constant was estimated to be 0.268 μM, was sensitive to decynium-22, an ENBT1 inhibitor, with the half maximal inhibitory concentration of 2.59 μM, which was comparable to that of 2.30 μM for [3H]adenine uptake by ENBT1 in its transient transfectant human embryonic kidney 293 cells. Although equilibrative nucleoside Transporter 1 (ENT1/SLC29A1) and ENT2/SLC29A2 are also known to be able to transport adenine, [3H]adenine uptake in HepG2 cells was not inhibited by the ENT1/2-specific inhibitor of either dipyridamole or nitrobenzylthioinosine. Finally, [3H]adenine uptake was extensively reduced by silencing of ENBT1 by RNA interference in the hepatocyte model. All these results, taken together, suggest the predominant role of ENBT1 in the uptake of adenine in HepG2 cells.
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Functional Analysis of the Role of Equilibrative Nucleobase Transporter 1 (ENBT1/SLC43A3) in Adenine Transport in HepG2 Cells
Journal of pharmaceutical sciences, 2020Co-Authors: Risa Takenaka, Katsuhisa Inoue, Tomoya Yasujima, Kinya Ohta, Junji Furukawa, Yosuke Hishikawa, Takahiro Yamashiro, Hiroaki YuasaAbstract:Abstract Equilibrative Nucleobase Transporter 1 (ENBT1/SLC43A3) has recently been identified as a purine-selective Nucleobase Transporter. Although it is highly expressed in the liver, its role in Nucleobase transport has not been confirmed yet in hepatocytes or any relevant cell models. We, therefore, examined its role in adenine transport in the HepG2 cell line as a human hepatocyte model. The uptake of [3H]adenine in HepG2 cells was highly saturable, indicating the involvement of carrier-mediated transport. The carrier-mediated transport component, for which the Michaelis constant was estimated to be 0.268 μM, was sensitive to decynium-22, an ENBT1 inhibitor, with the half maximal inhibitory concentration of 2.59 μM, which was comparable to that of 2.30 μM for [3H]adenine uptake by ENBT1 in its transient transfectant human embryonic kidney 293 cells. Although equilibrative nucleoside Transporter 1 (ENT1/SLC29A1) and ENT2/SLC29A2 are also known to be able to transport adenine, [3H]adenine uptake in HepG2 cells was not inhibited by the ENT1/2-specific inhibitor of either dipyridamole or nitrobenzylthioinosine. Finally, [3H]adenine uptake was extensively reduced by silencing of ENBT1 by RNA interference in the hepatocyte model. All these results, taken together, suggest the predominant role of ENBT1 in the uptake of adenine in HepG2 cells.
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Urate transport function of rat sodium-dependent Nucleobase Transporter 1.
Physiological reports, 2018Co-Authors: Tomoya Yasujima, Katsuhisa Inoue, Chihiro Murata, Yoshihisa Mimura, Tomoaki Murata, Masahiko Ohkubo, Kinya Ohta, Hiroaki YuasaAbstract:Sodium-dependent Nucleobase Transporter 1 (SNBT1) is a Nucleobase-specific Transporter identified in our recent study. In an attempt to search for its potential substrates other than Nucleobases in this study, we could successfully find urate, a metabolic derivative of purine Nucleobases, as a novel substrate, as indicated by its specific Na+ -dependent and saturable transport, with a Michaelis constant of 433 μmol/L, by rat SNBT1 (rSNBT1) stably expressed in Madin-Darby canine kidney II cells. However, urate uptake was observed only barely in the everted tissue sacs of the rat small intestine, in which rSNBT1 operates for Nucleobase uptake. These findings suggested that urate undergoes a futile cycle, in which urate transported into epithelial cells is immediately effluxed back by urate efflux Transporters, in the small intestine. In subsequent attempts to examine that possibility, such a futile urate cycle was demonstrated in the human embryonic kidney 293 cell line as a model cell system, where urate uptake induced by transiently introduced rSNBT1 was extensively reduced by the co-introduction of rat breast cancer resistance protein (rBCRP), a urate efflux Transporter present in the small intestine. However, urate uptake was not raised in the presence of Ko143, a BCRP inhibitor, in the everted intestinal tissue sacs, suggesting that some other Transporter might also be involved in urate efflux. The newly found urate transport function of SNBT1, together with the suggested futile urate cycle in the small intestine, should be of interest for its evolutional and biological implications, although SNBT1 is genetically deficient in humans.
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Dataset for: Urate transport function of rat sodium-dependent Nucleobase Transporter 1
2018Co-Authors: Tomoya Yasujima, Katsuhisa Inoue, Chihiro Murata, Yoshihisa Mimura, Tomoaki Murata, Masahiko Ohkubo, Kinya Ohta, Hiroaki YuasaAbstract:Sodium-dependent Nucleobase Transporter 1 (SNBT1) is a Nucleobase-specific Transporter identified in our recent study. In an attempt to search for its potential substrates other than Nucleobases in the present study, we could successfully find urate, a metabolic derivative of purine Nucleobases, as a novel substrate, as indicated by its specific Na+-dependent and saturable transport, with a Michaelis constant of 433 μM, by rat SNBT1 (rSNBT1) stably expressed in Madin-Darby canine kidney II cells. However, urate uptake was observed only barely in the everted tissue sacs of the rat small intestine, in which rSNBT1 operates for Nucleobase uptake. These findings suggested that urate undergoes a futile cycle, in which urate transported into epithelial cells is immediately effluxed back by urate efflux Transporters, in the small intestine. In subsequent attempts to examine that possibility, such a futile urate cycle was demonstrated in the human embryonic kidney 293 cell line as a model cell system, where urate uptake induced by transiently introduced rSNBT1 was extensively reduced by the co-introduction of rat breast cancer resistance protein (rBCRP), a urate efflux Transporter present in the small intestine. However, urate uptake was not raised in the presence of Ko143, a BCRP inhibitor, in the everted intestinal tissue sacs, suggesting that some other Transporter might also be involved in urate efflux. The newly found urate transport function of SNBT1, together with the suggested futile urate cycle in the small intestine, should be of interest for its evolutional and biological implications, although SNBT1 is genetically deficient in humans
Katsuhisa Inoue - One of the best experts on this subject based on the ideXlab platform.
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Current Understanding of the Intestinal Absorption of Nucleobases and Analogs.
Biological & pharmaceutical bulletin, 2020Co-Authors: Hiroaki Yuasa, Tomoya Yasujima, Katsuhisa InoueAbstract:It has long been suggested that a Na+-dependent carrier-mediated transport system is involved in the absorption of Nucleobases and analogs, including some drugs currently in therapeutic use, for their uptake at the brush border membrane of epithelial cells in the small intestine, mainly based on studies in non-primate experimental animals. The presence of this transport system was indeed proved by the recent identification of sodium-dependent Nucleobase Transporter 1 (SNBT1/Slc23a4) as its molecular entity in rats. However, this Transporter has been found to be genetically deficient in humans and higher primates. Aware of this deficiency, we need to revisit the issue of the absorption of these compounds in the human small intestine so that we can understand the mechanisms and gain information to assure the more rational use and development of drugs analogous to Nucleobases. Here, we review the current understanding of the intestinal absorption of Nucleobases and analogs. This includes recent knowledge about the efflux transport of those compounds across the basolateral membrane when exiting epithelial cells, following brush border uptake, in order to complete the overall absorption process; the facilitative Transporters of equilibrative nucleoside Transporter 1 (ENT1/SLC29A1) and equilibrative Nucleobase Transporter 1 (ENBT1/SLC43A3) may be involved in that in many animal species, including human and rat, without any major species differences.
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functional analysis of the role of equilibrative Nucleobase Transporter 1 enbt1 slc43a3 in adenine transport in hepg2 cells
Journal of Pharmaceutical Sciences, 2020Co-Authors: Risa Takenaka, Katsuhisa Inoue, Tomoya Yasujima, Kinya Ohta, Junji Furukawa, Yosuke Hishikawa, Takahiro Yamashiro, Hiroaki YuasaAbstract:Abstract Equilibrative Nucleobase Transporter 1 (ENBT1/SLC43A3) has recently been identified as a purine-selective Nucleobase Transporter. Although it is highly expressed in the liver, its role in Nucleobase transport has not been confirmed yet in hepatocytes or any relevant cell models. We, therefore, examined its role in adenine transport in the HepG2 cell line as a human hepatocyte model. The uptake of [3H]adenine in HepG2 cells was highly saturable, indicating the involvement of carrier-mediated transport. The carrier-mediated transport component, for which the Michaelis constant was estimated to be 0.268 μM, was sensitive to decynium-22, an ENBT1 inhibitor, with the half maximal inhibitory concentration of 2.59 μM, which was comparable to that of 2.30 μM for [3H]adenine uptake by ENBT1 in its transient transfectant human embryonic kidney 293 cells. Although equilibrative nucleoside Transporter 1 (ENT1/SLC29A1) and ENT2/SLC29A2 are also known to be able to transport adenine, [3H]adenine uptake in HepG2 cells was not inhibited by the ENT1/2-specific inhibitor of either dipyridamole or nitrobenzylthioinosine. Finally, [3H]adenine uptake was extensively reduced by silencing of ENBT1 by RNA interference in the hepatocyte model. All these results, taken together, suggest the predominant role of ENBT1 in the uptake of adenine in HepG2 cells.
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Functional Analysis of the Role of Equilibrative Nucleobase Transporter 1 (ENBT1/SLC43A3) in Adenine Transport in HepG2 Cells
Journal of pharmaceutical sciences, 2020Co-Authors: Risa Takenaka, Katsuhisa Inoue, Tomoya Yasujima, Kinya Ohta, Junji Furukawa, Yosuke Hishikawa, Takahiro Yamashiro, Hiroaki YuasaAbstract:Abstract Equilibrative Nucleobase Transporter 1 (ENBT1/SLC43A3) has recently been identified as a purine-selective Nucleobase Transporter. Although it is highly expressed in the liver, its role in Nucleobase transport has not been confirmed yet in hepatocytes or any relevant cell models. We, therefore, examined its role in adenine transport in the HepG2 cell line as a human hepatocyte model. The uptake of [3H]adenine in HepG2 cells was highly saturable, indicating the involvement of carrier-mediated transport. The carrier-mediated transport component, for which the Michaelis constant was estimated to be 0.268 μM, was sensitive to decynium-22, an ENBT1 inhibitor, with the half maximal inhibitory concentration of 2.59 μM, which was comparable to that of 2.30 μM for [3H]adenine uptake by ENBT1 in its transient transfectant human embryonic kidney 293 cells. Although equilibrative nucleoside Transporter 1 (ENT1/SLC29A1) and ENT2/SLC29A2 are also known to be able to transport adenine, [3H]adenine uptake in HepG2 cells was not inhibited by the ENT1/2-specific inhibitor of either dipyridamole or nitrobenzylthioinosine. Finally, [3H]adenine uptake was extensively reduced by silencing of ENBT1 by RNA interference in the hepatocyte model. All these results, taken together, suggest the predominant role of ENBT1 in the uptake of adenine in HepG2 cells.
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Urate transport function of rat sodium-dependent Nucleobase Transporter 1.
Physiological reports, 2018Co-Authors: Tomoya Yasujima, Katsuhisa Inoue, Chihiro Murata, Yoshihisa Mimura, Tomoaki Murata, Masahiko Ohkubo, Kinya Ohta, Hiroaki YuasaAbstract:Sodium-dependent Nucleobase Transporter 1 (SNBT1) is a Nucleobase-specific Transporter identified in our recent study. In an attempt to search for its potential substrates other than Nucleobases in this study, we could successfully find urate, a metabolic derivative of purine Nucleobases, as a novel substrate, as indicated by its specific Na+ -dependent and saturable transport, with a Michaelis constant of 433 μmol/L, by rat SNBT1 (rSNBT1) stably expressed in Madin-Darby canine kidney II cells. However, urate uptake was observed only barely in the everted tissue sacs of the rat small intestine, in which rSNBT1 operates for Nucleobase uptake. These findings suggested that urate undergoes a futile cycle, in which urate transported into epithelial cells is immediately effluxed back by urate efflux Transporters, in the small intestine. In subsequent attempts to examine that possibility, such a futile urate cycle was demonstrated in the human embryonic kidney 293 cell line as a model cell system, where urate uptake induced by transiently introduced rSNBT1 was extensively reduced by the co-introduction of rat breast cancer resistance protein (rBCRP), a urate efflux Transporter present in the small intestine. However, urate uptake was not raised in the presence of Ko143, a BCRP inhibitor, in the everted intestinal tissue sacs, suggesting that some other Transporter might also be involved in urate efflux. The newly found urate transport function of SNBT1, together with the suggested futile urate cycle in the small intestine, should be of interest for its evolutional and biological implications, although SNBT1 is genetically deficient in humans.
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Dataset for: Urate transport function of rat sodium-dependent Nucleobase Transporter 1
2018Co-Authors: Tomoya Yasujima, Katsuhisa Inoue, Chihiro Murata, Yoshihisa Mimura, Tomoaki Murata, Masahiko Ohkubo, Kinya Ohta, Hiroaki YuasaAbstract:Sodium-dependent Nucleobase Transporter 1 (SNBT1) is a Nucleobase-specific Transporter identified in our recent study. In an attempt to search for its potential substrates other than Nucleobases in the present study, we could successfully find urate, a metabolic derivative of purine Nucleobases, as a novel substrate, as indicated by its specific Na+-dependent and saturable transport, with a Michaelis constant of 433 μM, by rat SNBT1 (rSNBT1) stably expressed in Madin-Darby canine kidney II cells. However, urate uptake was observed only barely in the everted tissue sacs of the rat small intestine, in which rSNBT1 operates for Nucleobase uptake. These findings suggested that urate undergoes a futile cycle, in which urate transported into epithelial cells is immediately effluxed back by urate efflux Transporters, in the small intestine. In subsequent attempts to examine that possibility, such a futile urate cycle was demonstrated in the human embryonic kidney 293 cell line as a model cell system, where urate uptake induced by transiently introduced rSNBT1 was extensively reduced by the co-introduction of rat breast cancer resistance protein (rBCRP), a urate efflux Transporter present in the small intestine. However, urate uptake was not raised in the presence of Ko143, a BCRP inhibitor, in the everted intestinal tissue sacs, suggesting that some other Transporter might also be involved in urate efflux. The newly found urate transport function of SNBT1, together with the suggested futile urate cycle in the small intestine, should be of interest for its evolutional and biological implications, although SNBT1 is genetically deficient in humans
