The Experts below are selected from a list of 1422 Experts worldwide ranked by ideXlab platform
I. David Goldman - One of the best experts on this subject based on the ideXlab platform.
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Substituted-cysteine accessibility and cross-linking identify an exofacial cleft in the 7th and 8th helices of the proton-coupled Folate Transporter (SLC46A1)
American Journal of Physiology-cell Physiology, 2017Co-Authors: Srinivas Aluri, Rongbao Zhao, Andras Fiser, I. David GoldmanAbstract:The proton-coupled Folate Transporter (PCFT-SLC46A1) is required for Folate transport across the apical membrane of the small intestine and across the choroid plexus. This study focuses on the stru...
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Role of the tryptophan residues in proton-coupled Folate Transporter (PCFT-SLC46A1) function
American Journal of Physiology-cell Physiology, 2016Co-Authors: Mitra Najmi, Rongbao Zhao, Andras Fiser, I. David GoldmanAbstract:The proton-coupled Folate Transporter (PCFT) mediates Folate absorption across the brush-border membrane of the proximal small intestine and is required for Folate transport across the choroid plexus into the cerebrospinal fluid. In this study, the functional role and accessibility of the seven PCFT Trp residues were assessed by the substituted-cysteine accessibility method. Six Trp residues at a lipid-aqueous interface tolerated Cys substitution in terms of protein stability and function. W85C, W202C, and W213C were accessible to N-biotinyl aminoethylmethanethiosulfonate; W48C and W299C were accessible only after treatment with dithiotreitol (DTT), consistent with modification of these residues by an endogenous thiol-reacting molecule and their extracellular location. Neither W107C nor W333C was accessible (even after DTT) consistent with their cytoplasmic orientation. Biotinylation was blocked by pemetrexed only for the W48C (after DTT), W85C, W202C residues. Function was impaired only for the W299C PCF...
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Role of the tryptophan residues in proton-coupled Folate Transporter (PCFT-SLC46A1) function
American journal of physiology. Cell physiology, 2016Co-Authors: Mitra Najmi, Rongbao Zhao, Andras Fiser, I. David GoldmanAbstract:The proton-coupled Folate Transporter (PCFT) mediates Folate absorption across the brush-border membrane of the proximal small intestine and is required for Folate transport across the choroid plex...
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Identification of an Extracellular Gate for the Proton-coupled Folate Transporter (PCFT-SLC46A1) by Cysteine Cross-linking.
Journal of Biological Chemistry, 2016Co-Authors: Rongbao Zhao, Mitra Najmi, Andras Fiser, I. David GoldmanAbstract:Abstract The proton-coupled Folate Transporter (PCFT, SLC46A1) is required for intestinal Folate absorption and Folate homeostasis in humans. A homology model of PCFT, based upon the Escherichia coli glycerol 3-phosphate Transporter structure, predicted that PCFT transmembrane domains (TMDs) 1, 2, 7, and 11 form an extracellular gate in the inward-open conformation. To assess this model, five residues (Gln45-TMD1, Asn90-TMD2, Leu290-TMD7, Ser407-TMD11 and Asn411-TMD11) in the predicted gate were substituted with Cys to generate single and nine double mutants. Transport function of the mutants was assayed in transient transfectants by measurement of [3H]substrate influx as was accessibility of the Cys residues to biotinylation. Pairs of Cys residues were assessed for spontaneous formation of a disulfide bond, induction of a disulfide bond by oxidization with dichloro(1,10-phenanthroline)copper (II) (CuPh), or the formation of a Cd2+ complex. The data were consistent with the formation of a spontaneous disulfide bond between the N90C/S407C pair and a CuPh- and Cd2+-induced disulfide bond and complex, respectively, for the Q45C/L290C and L290C/N411C pairs. The decrease in activity induced by cross-linkage of the Cys residue pairs was due to a decrease in the influx Vmax consistent with restriction in the mobility of the Transporter. The presence of Folate substrate decreased the CuPh-induced inhibition of transport. Hence, the data support the glycerol 3-phosphate Transporter-based homology model of PCFT and the presence of an extracellular gate formed by TMDs 1, 2, 7, and 11.
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Identification of an Extracellular Gate for the Proton-coupled Folate Transporter (PCFT-SLC46A1) by Cysteine Cross-linking.
Journal of Biological Chemistry, 2016Co-Authors: Rongbao Zhao, Mitra Najmi, Andras Fiser, I. David GoldmanAbstract:Abstract The proton-coupled Folate Transporter (PCFT, SLC46A1) is required for intestinal Folate absorption and Folate homeostasis in humans. A homology model of PCFT, based upon the Escherichia coli glycerol 3-phosphate Transporter structure, predicted that PCFT transmembrane domains (TMDs) 1, 2, 7, and 11 form an extracellular gate in the inward-open conformation. To assess this model, five residues (Gln45-TMD1, Asn90-TMD2, Leu290-TMD7, Ser407-TMD11 and Asn411-TMD11) in the predicted gate were substituted with Cys to generate single and nine double mutants. Transport function of the mutants was assayed in transient transfectants by measurement of [3H]substrate influx as was accessibility of the Cys residues to biotinylation. Pairs of Cys residues were assessed for spontaneous formation of a disulfide bond, induction of a disulfide bond by oxidization with dichloro(1,10-phenanthroline)copper (II) (CuPh), or the formation of a Cd2+ complex. The data were consistent with the formation of a spontaneous disulfide bond between the N90C/S407C pair and a CuPh- and Cd2+-induced disulfide bond and complex, respectively, for the Q45C/L290C and L290C/N411C pairs. The decrease in activity induced by cross-linkage of the Cys residue pairs was due to a decrease in the influx Vmax consistent with restriction in the mobility of the Transporter. The presence of Folate substrate decreased the CuPh-induced inhibition of transport. Hence, the data support the glycerol 3-phosphate Transporter-based homology model of PCFT and the presence of an extracellular gate formed by TMDs 1, 2, 7, and 11.
Rongbao Zhao - One of the best experts on this subject based on the ideXlab platform.
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Substituted-cysteine accessibility and cross-linking identify an exofacial cleft in the 7th and 8th helices of the proton-coupled Folate Transporter (SLC46A1)
American Journal of Physiology-cell Physiology, 2017Co-Authors: Srinivas Aluri, Rongbao Zhao, Andras Fiser, I. David GoldmanAbstract:The proton-coupled Folate Transporter (PCFT-SLC46A1) is required for Folate transport across the apical membrane of the small intestine and across the choroid plexus. This study focuses on the stru...
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Role of the tryptophan residues in proton-coupled Folate Transporter (PCFT-SLC46A1) function
American Journal of Physiology-cell Physiology, 2016Co-Authors: Mitra Najmi, Rongbao Zhao, Andras Fiser, I. David GoldmanAbstract:The proton-coupled Folate Transporter (PCFT) mediates Folate absorption across the brush-border membrane of the proximal small intestine and is required for Folate transport across the choroid plexus into the cerebrospinal fluid. In this study, the functional role and accessibility of the seven PCFT Trp residues were assessed by the substituted-cysteine accessibility method. Six Trp residues at a lipid-aqueous interface tolerated Cys substitution in terms of protein stability and function. W85C, W202C, and W213C were accessible to N-biotinyl aminoethylmethanethiosulfonate; W48C and W299C were accessible only after treatment with dithiotreitol (DTT), consistent with modification of these residues by an endogenous thiol-reacting molecule and their extracellular location. Neither W107C nor W333C was accessible (even after DTT) consistent with their cytoplasmic orientation. Biotinylation was blocked by pemetrexed only for the W48C (after DTT), W85C, W202C residues. Function was impaired only for the W299C PCF...
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Role of the tryptophan residues in proton-coupled Folate Transporter (PCFT-SLC46A1) function
American journal of physiology. Cell physiology, 2016Co-Authors: Mitra Najmi, Rongbao Zhao, Andras Fiser, I. David GoldmanAbstract:The proton-coupled Folate Transporter (PCFT) mediates Folate absorption across the brush-border membrane of the proximal small intestine and is required for Folate transport across the choroid plex...
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Identification of an Extracellular Gate for the Proton-coupled Folate Transporter (PCFT-SLC46A1) by Cysteine Cross-linking.
Journal of Biological Chemistry, 2016Co-Authors: Rongbao Zhao, Mitra Najmi, Andras Fiser, I. David GoldmanAbstract:Abstract The proton-coupled Folate Transporter (PCFT, SLC46A1) is required for intestinal Folate absorption and Folate homeostasis in humans. A homology model of PCFT, based upon the Escherichia coli glycerol 3-phosphate Transporter structure, predicted that PCFT transmembrane domains (TMDs) 1, 2, 7, and 11 form an extracellular gate in the inward-open conformation. To assess this model, five residues (Gln45-TMD1, Asn90-TMD2, Leu290-TMD7, Ser407-TMD11 and Asn411-TMD11) in the predicted gate were substituted with Cys to generate single and nine double mutants. Transport function of the mutants was assayed in transient transfectants by measurement of [3H]substrate influx as was accessibility of the Cys residues to biotinylation. Pairs of Cys residues were assessed for spontaneous formation of a disulfide bond, induction of a disulfide bond by oxidization with dichloro(1,10-phenanthroline)copper (II) (CuPh), or the formation of a Cd2+ complex. The data were consistent with the formation of a spontaneous disulfide bond between the N90C/S407C pair and a CuPh- and Cd2+-induced disulfide bond and complex, respectively, for the Q45C/L290C and L290C/N411C pairs. The decrease in activity induced by cross-linkage of the Cys residue pairs was due to a decrease in the influx Vmax consistent with restriction in the mobility of the Transporter. The presence of Folate substrate decreased the CuPh-induced inhibition of transport. Hence, the data support the glycerol 3-phosphate Transporter-based homology model of PCFT and the presence of an extracellular gate formed by TMDs 1, 2, 7, and 11.
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Identification of an Extracellular Gate for the Proton-coupled Folate Transporter (PCFT-SLC46A1) by Cysteine Cross-linking.
Journal of Biological Chemistry, 2016Co-Authors: Rongbao Zhao, Mitra Najmi, Andras Fiser, I. David GoldmanAbstract:Abstract The proton-coupled Folate Transporter (PCFT, SLC46A1) is required for intestinal Folate absorption and Folate homeostasis in humans. A homology model of PCFT, based upon the Escherichia coli glycerol 3-phosphate Transporter structure, predicted that PCFT transmembrane domains (TMDs) 1, 2, 7, and 11 form an extracellular gate in the inward-open conformation. To assess this model, five residues (Gln45-TMD1, Asn90-TMD2, Leu290-TMD7, Ser407-TMD11 and Asn411-TMD11) in the predicted gate were substituted with Cys to generate single and nine double mutants. Transport function of the mutants was assayed in transient transfectants by measurement of [3H]substrate influx as was accessibility of the Cys residues to biotinylation. Pairs of Cys residues were assessed for spontaneous formation of a disulfide bond, induction of a disulfide bond by oxidization with dichloro(1,10-phenanthroline)copper (II) (CuPh), or the formation of a Cd2+ complex. The data were consistent with the formation of a spontaneous disulfide bond between the N90C/S407C pair and a CuPh- and Cd2+-induced disulfide bond and complex, respectively, for the Q45C/L290C and L290C/N411C pairs. The decrease in activity induced by cross-linkage of the Cys residue pairs was due to a decrease in the influx Vmax consistent with restriction in the mobility of the Transporter. The presence of Folate substrate decreased the CuPh-induced inhibition of transport. Hence, the data support the glycerol 3-phosphate Transporter-based homology model of PCFT and the presence of an extracellular gate formed by TMDs 1, 2, 7, and 11.
Michaela Jansen - One of the best experts on this subject based on the ideXlab platform.
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Expression in Sf9 insect cells, purification and functional reconstitution of the human proton-coupled Folate Transporter (PCFT, SLC46A1).
PLOS ONE, 2017Co-Authors: Swapneeta Date, Mariana C. Fiori, Guillermo A Altenberg, Michaela JansenAbstract:The proton-coupled Folate Transporter (PCFT) provides an essential uptake route for the vitamin folic acid (B9) in mammals. In addition, it is currently of high interest for targeting chemotherapeutic agents to tumors due to the increased folic acid requirement of rapidly dividing tumor cells as well as the upregulated PCFT expression in several tumors. To understand its function, determination of its atomic structure and molecular mechanism of transport are essential goals that require large amounts of functional PCFT. Here, we present a high-level heterologous expression system for human PCFT using a recombinant baculovirus and Spodoptera frugiperda (Sf9) insect cells. We demonstrate Folate transport functionality along the PCFT expression, isolation, and purification process. Importantly, purified PCFT transports folic acid after reconstitution. We thus succeeded in overcoming heterologous expression as a major bottleneck of PCFT research. The availability of an overexpression system for human PCFT provides the basis for future biochemical, biophysical and structural studies.
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Delineating the Extracellular Water-Accessible Surface of the Proton-Coupled Folate Transporter
2016Co-Authors: Phaneendra K Duddempudi, Raman Goyal, Swapneeta Sanjay Date, Michaela JansenAbstract:The proton-coupled Folate Transporter (PCFT) was recently identified as the major uptake route for dietary Folates in humans. The three-dimensional structure of PCFT and its detailed interplay with function remain to be determined. We screened the water-accessible extracellular surface of HsPCFT using the substituted-cysteine accessibility method, to investigate the boundaries between the water-accessible surface and inaccessible buried protein segments. Single-cysteines, engineered individually at 40 positions in a functional cysteine-less HsPCFT background construct, were probed for plasma-membrane expression in Xenopus oocytes with a bilayer-impermeant primary-amine-reactive biotinylating agent (sulfosuccinimidyl 6-(biotinamido) hexanoate), and additionally for water-accessibility of the respective engineered cysteine with the sulfhydryl-selective biotinylating agent 2-((biotinoyl)amino)ethyl methanethiosulfonate. The ratio between Cys-selective over amine-selective labeling was further used to evaluate three-dimensional models of HsPCFT generated by homology / threading modeling. The closest homologues of HsPCFT with a known experimentally-determined three-dimensional structure are all members of one of the largest membrane protein super-families, the major facilitator superfamily (MFS). The low sequence identity- 14 % or less – between HsPCFT and these templates necessitates experiment-based evaluation and model refinement of homology / threading models. With the present set of single-cysteine accessibilities, th
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Experimentally optimized threading structures of the proton-coupled Folate Transporter.
FEBS Open Bio, 2016Co-Authors: Swapneeta Date, Cheng-yen Charles Chen, Yidong Chen, Michaela JansenAbstract:The proton-coupled Folate Transporter (PCFT, SLC46A1) transports folic acid across the plasma membrane, together with an excess of protons such that the net charge translocation is positive. We developed 3D structural models of PCFT threaded onto the X-ray structures of major facilitator superfamily (MFS) members that were identified as close structural homologues. The model of PCFT threaded onto the glycerol-3-phosphate Transporter (GlpT) structure is consistent with detailed accessibility studies in the absence of extracellular substrate and at pH 7.4 presented here, and additionally with a multitude of other mutagenesis and functional studies. Characteristic MFS structural features are preserved in this PCFT model, such as 12 transmembrane helices divided into two pseudosymmetric bundles, and a high density of positive charges on the periphery of the cytoplasmic site that allow interactions with negatively charged lipid head-groups. Under the experimental conditions, PCFT predominantly samples the resting state, which in this case is inward-open. Several positions lining the substrate cavity have been identified. Motif A, a helix-turn-helix motif that is a hallmark of MFS Transporters between transmembrane segments II and III is oriented appropriately to interact with residues from transmembrane segments IV as well as XI upon conformational transition to the outward-open state. A charge-relay system between three charged residues as well as apposing glycines in two α-helices, both contributed to by motif A, become engaged when PCFT is modeled on the outward-open state of a putative proton-driven Transporter (YajR).
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Expression, Purification and Functional Characterization of Human Proton-Coupled Folate Transporter (SLC46A1)
Biophysical Journal, 2016Co-Authors: Swapneeta Date, Mariana C. Fiori, Ina L. Urbatsch, Michaela JansenAbstract:Folate cofactors play important roles in hundreds of metabolic reactions in a cell such as synthesis of protein and DNA precursors. The human proton-coupled Folate Transporter (PCFT) is the only means for absorption of dietary Folates. PCFT also transports anti-cancer agents such as Pemetrexed and Methotrexate. Inadequate supply of Folates and impaired PCFT function is associated with many disorders such as cancer, hereditary Folate malabsorption, hypercysteinemia, heart diseases, obesity, anemia, neural tube defects and Alzheimer's disease. It is therefore important to study structural and functional characteristics of PCFT, to understand Folate-homeostasis mechanisms and to develop better Folate-based therapies. The primary requirement in structural and functional characterization of mammalian membrane proteins such as PCFT is the availability of good expression systems and methods to obtain large quantities of purified protein. To address this primary concern, we are establishing an efficient system to over-express PCFT and developing methods to obtain purified PCFT in good yields. We have expressed human PCFT in insect, yeast, and bacterial cells. We have successfully purified PCFT to homogeneity. Importantly, we also demonstrate that the heterologously-expressed PCFT is functional. Our findings are significant because 12-transmembrane helical, mammalian membrane proteins such as PCFT are notoriously difficult to express in versatile prokaryotic and single-celled eukaryotic systems. Furthermore, due to their predominant hydrophobic character and requirement of detergent and/or lipid environment for their stability, such membrane proteins are difficult to purify. Each of the three PCFT expression systems that we have developed offers unique advantages over another depending on the intended use of PCFT. Our findings will be significant for the advancement of knowledge concerning both the structure and function of PCFT through basic science research as well as for industrial applications including design and screening of PCFT-targeted agents.
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Experimentally Defined Structural Model of the Human Proton-Coupled Folate Transporter
Biophysical Journal, 2014Co-Authors: Swapneeta Date, Michaela JansenAbstract:Folate cofactors, Vitamin B9, play crucial roles in more than a hundred one-carbon metabolism reactions in mammalian cells. Humans cannot synthesize Folates de novo and absorption through the diet is the only source of this vitamin. The proton-coupled Folate Transporter (PCFT) mediates this uptake in the upper small intestine by a pH-dependent process. PCFT also transports Folates into the central nervous system. Point mutations in the PCFT gene cause Hereditary Folate Malabsorption (HFM), with associated hematological and neurological defects due to impaired Folate transport. Certain solid tumor cell lines express high levels of PCFT mRNA. Importantly, the overall expression of PCFT is limited to only certain tissues in humans. Therefore, PCFT is a molecular target for specific delivery of anti-Folate chemotherapeutic agents to tumor cells. Unfortunately, the success rate of anti-Folate agents to reach clinical use is very low due to their side-effects. A structural model of PCFT can aid the development of specific anti-Folate agents for their PCFT-targeted delivery and thus minimize their side-effects. To optimize and verify an initial structural model of PCFT, we applied a wide array of experimental approaches: solvent accessibility profiling through substituted cysteine accessibility scanning, studying the helix packing, and assaying the functionality of PCFT mutants. We complemented the experimental data with theoretical approaches for structure prediction such as homology modeling/threading, ligand docking and an extensive review of other secondary Transporters and Folate transport proteins. With these combined approaches, we have developed a structural model of PCFT that accurately reflects our experimental observations. This model forms a basis to understand the impacts of HFM mutations, and to develop Folate analogues for Folate deficiency intervention. Additionally, it will aid the design of PCFT structure-based anti-Folate agents for the treatment of cancer.
Larry H Matherly - One of the best experts on this subject based on the ideXlab platform.
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Folate Transporter dynamics and therapy with classic and tumor-targeted antiFolates.
Scientific reports, 2021Co-Authors: Carrie O'connor, Adrianne Wallace-povirk, Aleem Gangjee, Larry H Matherly, Josephine Frühauf, Changwen Ning, Nian Tong, Zhanjun HouAbstract:There are three major Folate uptake systems in human tissues and tumors, including the reduced Folate carrier (RFC), Folate receptors (FRs) and proton-coupled Folate Transporter (PCFT). We studied the functional interrelationships among these systems for the novel tumor-targeted antiFolates AGF94 (transported by PCFT and FRs but not RFC) and AGF102 (selective for FRs) versus the classic antiFolates pemetrexed, methotrexate and PT523 (variously transported by FRs, PCFT and RFC). We engineered HeLa cell models to express FRα or RFC under control of a tetracycline-inducible promoter with or without constitutive PCFT. We showed that cellular accumulations of extracellular Folates were determined by the type and levels of the major Folate Transporters, with PCFT and RFC prevailing over FRα, depending on expression levels and pH. Based on patterns of cell proliferation in the presence of the inhibitors, we established transport redundancy for RFC and PCFT in pemetrexed uptake, and for PCFT and FRα in AGF94 uptake; uptake by PCFT predominated for pemetrexed and FRα for AGF94. For methotrexate and PT523, uptake by RFC predominated even in the presence of PCFT or FRα. For both classic (methotrexate, PT523) and FRα-targeted (AGF102) antiFolates, anti-proliferative activities were antagonized by PCFT, likely due to its robust activity in mediating Folate accumulation. Collectively, our findings describe a previously unrecognized interplay among the major Folate transport systems that depends on Transporter levels and extracellular pH, and that determines their contributions to the uptake and anti-tumor efficacies of targeted and untargeted antiFolates.
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Development and validation of chemical features-based proton-coupled Folate Transporter/activity and reduced Folate carrier/activity models (pharmacophores).
Journal of Molecular Graphics & Modelling, 2018Co-Authors: Khushbu Shah, Sudhir Raghavan, Larry H Matherly, Aleem GangjeeAbstract:Abstract All clinically used antiFolates lack transport selectivity for tumors over normal cells resulting in dose-limiting toxicities. There is growing interest in developing novel tumor-targeted cytotoxic antiFolates with selective transport into tumors over normal cells via the proton-coupled Folate Transporter (PCFT) over the ubiquitously expressed reduced Folate carrier (RFC). A lack of X-ray crystal structures or predictive models for PCFT or RFC has hindered structure-aided drug design for PCFT-selective therapeutics. Four-point validated models (pharmacophores) were generated for PCFT/Activity (HBA, NI, RA, RA) and RFC/Activity (HBD, NI, HBA, HBA) based on inhibition (IC50) of proliferation of isogenic Chinese hamster ovary (CHO) cells engineered to express only human PCFT or only RFC. Our results revealed substantial differences in structural features required for transport of novel molecules by these Transporters which can be utilized for developing Transporter-selective antiFolates.
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development and validation of chemical features based proton coupled Folate Transporter activity and reduced Folate carrier activity models pharmacophores
Journal of Molecular Graphics & Modelling, 2018Co-Authors: Khushbu Shah, Sudhir Raghavan, Larry H Matherly, Zhanjun Hou, Aleem GangjeeAbstract:Abstract All clinically used antiFolates lack transport selectivity for tumors over normal cells resulting in dose-limiting toxicities. There is growing interest in developing novel tumor-targeted cytotoxic antiFolates with selective transport into tumors over normal cells via the proton-coupled Folate Transporter (PCFT) over the ubiquitously expressed reduced Folate carrier (RFC). A lack of X-ray crystal structures or predictive models for PCFT or RFC has hindered structure-aided drug design for PCFT-selective therapeutics. Four-point validated models (pharmacophores) were generated for PCFT/Activity (HBA, NI, RA, RA) and RFC/Activity (HBD, NI, HBA, HBA) based on inhibition (IC50) of proliferation of isogenic Chinese hamster ovary (CHO) cells engineered to express only human PCFT or only RFC. Our results revealed substantial differences in structural features required for transport of novel molecules by these Transporters which can be utilized for developing Transporter-selective antiFolates.
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The promise and challenges of exploiting the proton-coupled Folate Transporter for selective therapeutic targeting of cancer
Cancer Chemotherapy and Pharmacology, 2018Co-Authors: Larry H Matherly, Aleem GangjeeAbstract:This review considers the “promise” of exploiting the proton-coupled Folate Transporter (PCFT) for selective therapeutic targeting of cancer. PCFT was discovered in 2006 and was identified as the principal Folate Transporter involved in the intestinal absorption of dietary Folates. The recognition that PCFT was highly expressed in many tumors stimulated substantial interest in using PCFT for cytotoxic drug targeting, taking advantage of its high level transport activity under the acidic pH conditions that characterize many tumors. For pemetrexed, among the best PCFT substrates, transport by PCFT establishes its importance as a clinically important Transporter in malignant pleural mesothelioma and non-small cell lung cancer. In recent years, the notion of PCFT-targeting has been extended to a new generation of tumor-targeted 6-substituted pyrrolo[2,3- d ]pyrimidine compounds that are structurally and functionally distinct from pemetrexed, and that exhibit near exclusive transport by PCFT and potent inhibition of de novo purine nucleotide biosynthesis. Based on compelling preclinical evidence in a wide range of human tumor models, it is now time to advance the most optimized PCFT-targeted agents with the best balance of PCFT transport specificity and potent antitumor efficacy to the clinic to validate this novel paradigm of highly selective tumor targeting.
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Functional and mechanistic roles of the human proton-coupled Folate Transporter transmembrane domain 6-7 linker
Biochemical Journal, 2016Co-Authors: M. Roy Wilson, Lucas J. Wilson, Jun Ye, Larry H MatherlyAbstract:The proton-coupled Folate Transporter (PCFT; SLC46A1) is a Folate-proton symporter expressed in solid tumors and is used for tumor-targeted delivery of cytotoxic antiFolates. Topology modeling suggests that PCFT secondary structure includes twelve transmembrane domains (TMDs) with TMDs 6 and 7 linked by an intracellular loop (positions 236 to 265) including His247, implicated as functionally important. Single cysteine mutants were inserted from positions 241 to 251 in cysteine-less PCFT and mutant proteins were expressed in PCFT-null (R1-11) HeLa cells; none were reactive with 2-aminoethyl methanethiosulfonate biotin, suggesting that the TMD6-7 loop is intracellular. Twenty-nine single alanine mutants spanning the entire TMD6-7 loop were expressed in R1-11 cells; activity was generally preserved, with the exception of the 247, 250 and 251 mutants, partly due to decreased surface expression. Co-expression of PCFT TMD1-6 and TMD7-12 half-molecules in R1-11 cells partially restored transport activity, although removal of residues 252-265 from TMD7-12 abolished transport. Chimeric proteins, including non-homologous sequence from a thiamine Transporter (ThTr1) inserted into the PCFT TMD6-7 loop (positions 236 to 250, or 251 to 265), were active, although replacement of the entire loop with ThTr1 sequence resulted in substantial loss of activity. Amino acid replacements (Ala, Arg, His, Gln, Glu) or deletions at position 247 in wild-type and PCFT-ThTr1 chimeras resulted in differential effects on transport. Collectively, our findings suggest that the PCFT TMD6-7 connecting loop confers protein stability and may serve a unique functional role that depends on secondary structure rather than particular sequence elements.
Hiroaki Yuasa - One of the best experts on this subject based on the ideXlab platform.
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Identification of the amino acid residue responsible for the myricetin sensitivity of human proton-coupled Folate Transporter
Scientific Reports, 2019Co-Authors: Takahiro Yamashiro, Tomoya Yasujima, Kinya Ohta, Katsuhisa Inoue, Hiroaki YuasaAbstract:Human proton-coupled Folate Transporter (hPCFT/SLC46A1) has recently been found to be inhibited by myricetin by a sustained mechanism, raising a concern that the inhibition might lead to malabsorption of Folates in the intestine, where hPCFT works for their epithelial uptake. However, rat PCFT (rPCFT) has more recently been found not to be inhibited by myricetin. Prompted by this finding, we attempted to determine the amino acid residue involved in that by analyses comparing between hPCFT and rPCFT. In the initial analysis, chimeric constructs prepared from hPCFT and rPCFT were examined for myricetin sensitivity to determine the hPCFT segment involved in the sensitivity. Focusing on the thereby determined segment from 83rd to 186th amino acid residue, hPCFT mutants having a designated amino acid residue replaced with its counterpart in rPCFT were prepared for the subsequent analysis. Among them, only G158N-substituted hPCFT was found to be transformed to be insensitive to myricetin and, accordingly, oppositely N158G-substituted rPCFT was transformed to be sensitive to myricetin. These results indicate the critical role of Gly^158 in the myricetin sensitivity of hPCFT. This finding would help advance the elucidation of the mechanism of the myricetin-induced inhibition of hPCFT and manage the potential risk arising from that.
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Specific inhibitory effects of myricetin on human proton-coupled Folate Transporter: Comparison with its effects on rat proton-coupled Folate Transporter and human riboflavin Transporter 3.
Drug Metabolism and Pharmacokinetics, 2017Co-Authors: Takahiro Yamashiro, Tomoya Yasujima, Kinya Ohta, Katsuhisa Inoue, Hiroaki YuasaAbstract:Abstract Myricetin is a flavonoid that inhibits human proton-coupled Folate Transporter (hPCFT) in a transient manner, in which inhibition is manifested in its presence, and also in a sustained manner, in which inhibition induced in its presence persists after its removal. In an effort to elucidate the mechanisms involved in those, we examined if myricetin might or might not act similarly on some other Transporters. Transporters examined for that, in comparison with hPCFT, were its rat ortholog (rPCFT) and human riboflavin Transporter 3 (hRFVT3). Experiments were conducted, using human embryonic kidney 293 cells transiently expressing the Transporter to be examined, to assess the effects of myricetin (100 μM) on the uptake of Folate by the PCFTs and riboflavin by hRFVT3. For hPCFT, myricetin was confirmed to induce a transient inhibition and also a sustained inhibition. However, myricetin induced neither transient nor sustained type of rPCFT inhibition. hRFVT3 was inhibited by myricetin in a transient manner, but not in a sustained manner. These results suggest the involvement of a hPCFT-specific mechanism in the sustained inhibition. The transient inhibition may be induced by a mechanism specific to hPCFT and also hRFVT3.
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Kinetic and time-dependent features of sustained inhibitory effect of myricetin on Folate transport by proton-coupled Folate Transporter.
Drug Metabolism and Pharmacokinetics, 2015Co-Authors: Takahiro Yamashiro, Kinya Ohta, Yayoi Hayashi, Mai Furumiya, Katsuhisa Inoue, Hiroaki YuasaAbstract:Abstract Myricetin is a flavonoid that has recently been suggested to induce sustained inhibition of proton-coupled Folate Transporter (PCFT/SLC46A1), which operates for intestinal Folate uptake. The present study was conducted to characterize the inhibitory effect in more detail, using human PCFT stably expressed in Madin–Darby canine kidney II cells, to gain information to cope with problems potentially arising from that. The kinetics of saturable Folate transport was first assessed in the absence of myricetin in the cells pretreated with the flavonoid for 60 min. The pretreatment induced PCFT inhibition in a manner dependent on the concentration of myricetin, where the maximum transport rate was reduced by 35.5% and 83.1%, respectively, at its concentrations of 20 μM and 50 μM. The inhibitory effect was, however, less extensive at lower Folate concentrations, because the Michaelis constant was also reduced similarly in a manner dependent on myricetin concentration. The inhibition was induced depending on the time of pretreatment and, after removal of myricetin (50 μM) upon the manifestation of an extensive inhibition at 60 min, reversed almost completely in 90 min. This rather short time required for recovery may suggest that the sustained inhibition of PCFT is of a reversible type.
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Sustained inhibition of proton-coupled Folate Transporter by myricetin
Drug Metabolism and Pharmacokinetics, 2014Co-Authors: Mai Furumiya, Takahiro Yamashiro, Kinya Ohta, Yayoi Hayashi, Katsuhisa Inoue, Chihiro Nishijima, Hiroaki YuasaAbstract:Summary Myricetin is a flavonoid that has recently been suggested to interfere with the intestinal Folate transport system. To examine that possibility, focusing on its sustained inhibitory effect on proton-coupled Folate Transporter (PCFT), the uptake of Folate was examined in Caco-2 cells, in which PCFT is known to be in operation, in the absence of myricetin in the medium during uptake period after preincubation of the cells with the flavonoid (100 μM) for 1 h. This pretreatment induced an extensive and sustained reduction in the carrier-mediated component of Folate uptake, which was attributable to a reduction in the maximum transport rate ( V max ). Although the affinity of the Transporter for Folate was increased at the same time as indicated by a reduction in the Michaelis constant ( K m ), the change in K m was overwhelmed in extent by that in V max . Consistent with the finding, Folate transport by human PCFT stably expressed in Madin–Darby canine kidney II cells was reduced in a similar manner with simultaneous reductions in V max and K m by myricetin pretreatment. Attention may need to be given for a possibility that such a sustained inhibition of PCFT could potentially be a cause of the malabsorption of Folate and also antiFolate drugs.
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Noncompetitive Inhibition of Proton-coupled Folate Transporter by Myricetin
Drug Metabolism and Pharmacokinetics, 2014Co-Authors: Mai Furumiya, Takahiro Yamashiro, Kinya Ohta, Yayoi Hayashi, Erina Inaoka, Katsuhisa Inoue, Chihiro Nishijima, Hiroaki YuasaAbstract:Summary: Myricetin is a flavonoid that has recently been suggested to interfere with the intestinal Folate transport system. The present study was conducted to examine that possibility, focusing on its inhibitory effect on proton-coupled Folate Transporter (PCFT) as the molecular entity of the transport system. The uptake transport of Folate was first examined in the Caco-2 cell as an intestinal epithelial cell model, and its carrier-mediated component, of which the Michaelis constant ( K m ) was 0.407 μΜ, was found to be noncompetitively inhibited by myricetin with an inhibition constant ( K i ) of 61 μΜ. Consistent with that, Folate transport by human PCFT stably expressed in Madin-Darby canine kidney II (MDCKII) cells, of which the K m was 1.246 μM, was also noncompetitively inhibited by myricetin with a K i of 130 μM. Thus, myricetin was suggested to inhibit intestinal Folate transport by acting noncompetitively on PCFT, although the K m and K i were similarly shifted to some extent to be smaller in Caco-2 cells. Finally, epigallocatechin-3-gallate was also suggested to act in a noncompetitive manner as an inhibitory flavonoid. Care may need to be taken, therefore, in the ingestion of myricetin and some flavonoids to maintain the absorption of Folate and antiFolate drugs.