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Thierry Pourcher - One of the best experts on this subject based on the ideXlab platform.
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the sodium iodide Symporter state of the art of its molecular characterization
Biochimica et Biophysica Acta, 2014Co-Authors: Elisabeth Darrouzet, Sabine Lindenthal, Didier Marcellin, Jeanluc Pellequer, Thierry PourcherAbstract:The sodium/iodide Symporter (NIS or SLC5A5) is an intrinsic membrane protein implicated in iodide uptake into thyroid follicular cells. It plays a crucial role in iodine metabolism and thyroid regulation and its function is widely exploited in the diagnosis and treatment of benign and malignant thyroid diseases. A great effort is currently being made to develop a NIS-based gene therapy also allowing the radiotreatment of nonthyroidal tumors. NIS is also expressed in other tissues, such as salivary gland, stomach and mammary gland during lactation, where its physiological role remains unclear. The molecular identity of the thyroid iodide transporter was elucidated approximately fifteen years ago. It belongs to the superfamily of sodium/solute Symporters, SSS (and to the human transporter family, SLC5), and is composed of 13 transmembrane helices and 643 amino acid residues in humans. Knowledge concerning NIS structure/function relationship has been obtained by taking advantage of the high resolution structure of one member of the SSS family, the Vibrio parahaemolyticus sodium/galactose Symporter (vSGLT), and from studies of gene mutations leading to congenital iodine transport defects (ITD). This review will summarize current knowledge regarding the molecular characterization of NIS.
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The sodium/iodide Symporter: State of the art of its molecular characterization
Biochimica et Biophysica Acta:Biomembranes, 2014Co-Authors: Elisabeth Darrouzet, Sabine Lindenthal, Didier Marcellin, Jeanluc Pellequer, Thierry PourcherAbstract:The sodium/iodide Symporter (NIS or SLC5A5) is an intrinsic membrane protein implicated in iodide uptake into thyroid follicular cells. It plays a crucial role in iodine metabolism and thyroid regulation and its function is widely exploited in the diagnosis and treatment of benign and malignant thyroid diseases. A great effort is currently being made to develop a NIS-based gene therapy also allowing the radiotreatment of nonthyroidal tumors. NIS is also expressed in other tissues, such as salivary gland, stomach and mammary gland during lactation, where its physiological role remains unclear. The molecular identity of the thyroid iodide transporter was elucidated approximately fifteen years ago. It belongs to the superfamily of sodium/solute Symporters, SSS (and to the human transporter family, SLC5), and is composed of 13 transmembrane helices and 643 amino acid residues in humans. Knowledge concerning NIS structure/function relationship has been obtained by taking advantage of the high resolution structure of one member of the SSS family, the Vibrio parahaemolyticus sodium/galactose Symporter (vSGLT), and from studies of gene mutations leading to congenital iodine transport defects (ITD). This review will summarize current knowledge regarding the molecular characterization of NIS.
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distribution and dynamics of 99mtc pertechnetate uptake in the thyroid and other organs assessed by single photon emission computed tomography in living mice
Thyroid, 2010Co-Authors: Philippe R. Franken, Julien Guglielmi, Malick Koulibaly, Michel Defrise, Jacques Darcourt, Christian Vanhove, Thierry PourcherAbstract:Background: 99mTc pertechnetate is a well-known anion, used for clinical imaging of thyroid function. This gamma emitter is transported by the sodium iodide Symporter but is not incorporated into thyroglobulin. Scintigraphy using 99mTc pertechnetate or 123iodide represents a powerful tool for the study of sodium iodide Symporter activity in different organs of living animal models. However, in many studies that have been performed in mice, the thyroid could not be distinguished from the salivary glands. In this work, we have evaluated the use of a clinically dedicated single-photon emission computed tomography (SPECT) camera for thyroid imaging and assessed what improvements are necessary for the development of this technique. Methods: SPECT of the mouse neck region, with pinhole collimation and geometric calibration, was used for the individual measurement of 99mTc pertechnetate uptake in the thyroid and the salivary glands. Uptake in the stomach was studied by planar whole-body imaging. Uptake kinetics ...
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a 96 well automated radioiodide uptake assay for sodium iodide Symporter inhibitors
Assay and Drug Development Technologies, 2007Co-Authors: Nathalie Lecatguillet, Thierry Pourcher, Goulven Merer, Roman Lopez, Bernard Rousseau, Yves AmbroiseAbstract:A high-throughput screening method based on radioiodide uptake in human embryonic kidney 293 cells expressing the human sodium/iodide Symporter was developed. Central to assay development was a homogeneous cell culture in the 96-well microplate coupled with the use of scintillation proximity technology. The assay is fast and highly reproducible with a Z′ greater than 0.8. The automated procedure allows the screening of 4,000 compounds per day. Using this methodology, several known substrates of the sodium/iodide Symporter were evaluated in a single day. Inhibition of iodide uptake was shown to follow the series PF6− > ClO4− > BF4− > SCN− >> NO3− > IO4− > N3− >> Br−, in accord with the literature. This method represents an initial approach to the search for inhibitors of iodide transport mediated by the sodium/iodide Symporter.
Christine Ziegler - One of the best experts on this subject based on the ideXlab platform.
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substrate bound outward open state of the betaine transporter betp provides insights into na coupling
Nature Communications, 2014Co-Authors: Camilo Perez, Christine Ziegler, Belinda Faust, Ahmad Reza Mehdipour, Kevin A. FrancesconiAbstract:The Na(+)-coupled betaine Symporter BetP shares a highly conserved fold with other sequence unrelated secondary transporters, for example, with neurotransmitter Symporters. Recently, we obtained atomic structures of BetP in distinct conformational states, which elucidated parts of its alternating-access mechanism. Here, we report a structure of BetP in a new outward-open state in complex with an anomalous scattering substrate, adding a fundamental piece to an unprecedented set of structural snapshots for a secondary transporter. In combination with molecular dynamics simulations these structural data highlight important features of the sequential formation of the substrate and sodium-binding sites, in which coordinating water molecules play a crucial role. We observe a strictly interdependent binding of betaine and sodium ions during the coupling process. All three sites undergo progressive reshaping and dehydration during the alternating-access cycle, with the most optimal coordination of all substrates found in the closed state.
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role of bundle helices in a regulatory crosstalk in the trimeric betaine transporter betp
Journal of Molecular Biology, 2011Co-Authors: Rebecca M Gartner, Camilo Perez, Caroline Koshy, Christine ZieglerAbstract:The Na(+)-coupled betaine Symporter BetP regulates transport activity in response to hyperosmotic stress only in its trimeric state, suggesting a regulatory crosstalk between individual protomers. BetP shares the overall fold of two inverted structurally related five-transmembrane (TM) helix repeats with the sequence-unrelated Na(+)-coupled Symporters LeuT, vSGLT, and Mhp1, which are neither trimeric nor regulated in transport activity. Conformational changes characteristic for this transporter fold involve the two first helices of each repeat, which form a four-TM-helix bundle. Here, we identify two ionic networks in BetP located on both sides of the membrane that might be responsible for BetP's unique regulatory behavior by restricting the conformational flexibility of the four-TM-helix bundle. The cytoplasmic ionic interaction network links both first helices of each repeat in one protomer to the osmosensing C-terminal domain of the adjacent protomer. Moreover, the periplasmic ionic interaction network conformationally locks the four-TM-helix bundle between the same neighbor protomers. By a combination of site-directed mutagenesis, cross-linking, and betaine uptake measurements, we demonstrate how conformational changes in individual bundle helices are transduced to the entire bundle by specific inter-helical interactions. We suggest that one purpose of bundle networking is to assist crosstalk between protomers during transport regulation by specifically modulating the transition from outward-facing to inward-facing state.
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molecular basis of transport and regulation in the na betaine Symporter betp
Nature, 2009Co-Authors: Susanne Ressl, Anke Terwisscha C Van Scheltinga, Clemens Vonrhein, Vera Ott, Christine ZieglerAbstract:Osmoregulated transporters sense intracellular osmotic pressure and respond to hyperosmotic stress by accumulation of osmolytes to restore normal hydration levels. Here we report the determination of the X-ray structure of a member of the family of betaine/choline/carnitine transporters, the Na+-coupled Symporter BetP from Corynebacterium glutamicum, which is a highly effective osmoregulated uptake system for glycine betaine. Glycine betaine is bound in a tryptophan box occluded from both sides of the membrane with aromatic side chains lining the transport pathway. BetP has the same overall fold as three unrelated Na+-coupled Symporters. Whereas these are crystallized in either the outward-facing or the inward-facing conformation, the BetP structure reveals a unique intermediate conformation in the Na+-coupled transport cycle. The trimeric architecture of BetP and the break in three-fold symmetry by the osmosensing C-terminal helices suggest a regulatory mechanism of Na+-coupled osmolyte transport to counteract osmotic stress. Microorganisms need to adapt rapidly to extreme variations in salinity, temperature or osmolarity. Many of them counteract high osmolarity via the intracellular accumulation of osmolytes, highly polar, organic compounds that promote protein stability through unfavourable interactions with the unfolded state. High-affinity transporters are used to import osmolytes from extracellular sources and Ressl et al. report the determination of the X-ray structure of a member of the family of betaine/choline/carnitine transporters, the Na+-coupled Symporter BetP, which is a highly effective osmoregulated uptake system for glycine betaine. The crystal structure shows BetP to have the same overall fold as other, unrelated Na+-coupled transporters, but it is captured in a unique intermediate conformation whereby the glycine betaine substrate is occluded from both sides of the membrane. The structure of BetP therefore provides new insights into both osmoregulation and the fundamental mechanisms of membrane transport. This paper presents the first X-ray structure of a member of the the betaine/choline/carnitine transporter family. This Na+-coupled Symporter (BetP) has the same overall fold as other unrelated Na+-coupled transporters and is captured in an intermediate conformation, whereby the substrate (glycine betaine) is occluded from both sides of the membrane.
Maria Jose Leandro - One of the best experts on this subject based on the ideXlab platform.
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characterization of new polyol h Symporters in debaryomyces hansenii
PLOS ONE, 2014Co-Authors: Iliana Pereira, Maria C Loureirodias, Catarina Prista, Ana Carla Madeira, Maria Jose LeandroAbstract:Debaryomyces hansenii is a halotolerant yeast that produces and assimilates a wide variety of polyols. In this work we evaluate polyol transport in D. hansenii CBS 767, detecting the occurrence of polyol/H+ (and sugar/H+) Symporter activity, through the transient extracellular alkalinization of unbuffered starved cell suspensions. From the D. hansenii genome database, we selected nine ORFs encoding putative transporter proteins to clone in a centromeric plasmid with C-terminal GFP tagging and screened for polyol/H+ Symporters by heterologous expression in Saccharomyces cerevisiae. Five distinct D. hansenii polyol/H+ Symporters were identified and characterized, with different specificities and affinities for polyols, namely one glycerol-specific (DhStl1), one D-galactitol-specific (DhSgl1, Symporter galactitol/H+ 1), one D-(+)-chiro-inositol-specific (DhSyi1, Symporter D-(+)-chiro-inositol/H+ 1), one for D-sorbitol/D-mannitol/ribitol/D-arabitol/D-galactitol (DhSyl1, Symporter Polyols 1) and another for D-sorbitol/D-mannitol/ribitol/D-arabitol (DhSyl2, Symporter Polyols 2). This work contributed to the annotation of new yeast polyol transporters, including two specific for uncommon substrates as galactitol and D-(+)-chiro-inositol.
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Characterization of New Polyol/H+ Symporters in Debaryomyces hansenii
2014Co-Authors: Iliana Pereira, Maria C. Loureiro-dias, Catarina Prista, Ana Madeira, Maria Jose LeandroAbstract:Debaryomyces hansenii is a halotolerant yeast that produces and assimilates a wide variety of polyols. In this work we evaluate polyol transport in D. hansenii CBS 767, detecting the occurrence of polyol/H+ (and sugar/H+) Symporter activity, through the transient extracellular alkalinization of unbuffered starved cell suspensions. From the D. hansenii genome database, we selected nine ORFs encoding putative transporter proteins to clone in a centromeric plasmid with C-terminal GFP tagging and screened for polyol/H+ Symporters by heterologous expression in Saccharomyces cerevisiae. Five distinct D. hansenii polyol/H+ Symporters were identified and characterized, with different specificities and affinities for polyols, namely one glycerol-specific (DhStl1), one D-galactitol-specific (DhSgl1, Symporter galactitol/H+ 1), one D-(+)-chiro-inositol-specific (DhSyi1, Symporter D-(+)-chiro-inositol/H+ 1), one for D-sorbitol/D-mannitol/ribitol/D-arabitol/D-galactitol (DhSyl1, Symporter Polyols 1) and another for D-sorbitol/D-mannitol/ribitol/D-arabitol (DhSyl2, Symporter Polyols 2). This work contributed to the annotation of new yeast polyol transporters, including two specific for uncommon substrates as galactitol and D-(+)-chiro-inositol.
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zrfsy1 a high affinity fructose h Symporter from fructophilic yeast zygosaccharomyces rouxii
PLOS ONE, 2013Co-Authors: Maria Jose Leandro, Hana Sychrová, Catarina Prista, Maria C LoureirodiasAbstract:Zygosaccharomyces rouxii is a fructophilic yeast than can grow at very high sugar concentrations. We have identified an ORF encoding a putative fructose/H+ Symporter in the Z. rouxii CBS 732 genome database. Heterologous expression of this ORF in a S. cerevisiae strain lacking its own hexose transporters (hxt-null) and subsequent kinetic characterization of its sugar transport activity showed it is a high-affinity low-capacity fructose/H+ Symporter, with Km 0.45±0.07 mM and Vmax 0.57±0.02 mmol h−1 (gdw) −1. We named it ZrFsy1. This protein also weakly transports xylitol and sorbose, but not glucose or other hexoses. The expression of ZrFSY1 in Z. rouxii is higher when the cells are cultivated at extremely low fructose concentrations (<0.2%) and on non-fermentable carbon sources such as mannitol and xylitol, where the cells have a prolonged lag phase, longer duplication times and change their microscopic morphology. A clear phenotype was determined for the first time for the deletion of a fructose/H+ Symporter in the genome where it occurs naturally. The effect of the deletion of ZrFSY1 in Z. rouxii cells is only evident when the cells are cultivated at very low fructose concentrations, when the ZrFsy1 fructose Symporter is the main active fructose transporter system.
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two glucose xylose transporter genes from the yeast candida intermedia first molecular characterization of a yeast xylose h Symporter
Biochemical Journal, 2006Co-Authors: Maria Jose Leandro, Paula Gonçalves, Isabel SpencermartinsAbstract:Candida intermedia PYCC 4715 was previously shown to grow well on xylose and to transport this sugar by two different transport systems: high-capacity and low-affinity facilitated diffusion and a high-affinity xylose–proton Symporter, both of which accept glucose as a substrate. Here we report the isolation of genes encoding both transporters, designated GXF1 (glucose/xylose facilitator 1) and GXS1 (glucose/xylose Symporter 1) respectively. Although GXF1 was isolated by functional complementation of an HXT-null (where Hxt refers to hexose transporters) Saccharomyces cerevisiae strain, isolation of the GXS1 cDNA required partial purification and micro-sequencing of the transporter, identified by its relative abundance in cells grown on low xylose concentrations. Both genes were expressed in S. cerevisiae and the kinetic parameters of glucose and xylose transport were determined. Gxs1 is the first yeast xylose/glucose–H+ Symporter to be characterized at the molecular level. Comparison of its amino acid sequence with available sequence data revealed the existence of a family of putative monosaccharide–H+ Symporters encompassing proteins from several yeasts and filamentous fungi.
Tsuneo Saga - One of the best experts on this subject based on the ideXlab platform.
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single photon emission computed tomography of spontaneous liver metastasis from orthotopically implanted human colon cancer cell line stably expressing human sodium iodide Symporter reporter gene
EJNMMI research, 2012Co-Authors: Masayuki Inubushi, Chika Murai, Hironobu Hata, Yoshimasa Kitagawa, Tsuneo SagaAbstract:Background We aimed to develop a mouse spontaneous liver metastasis model from an orthotopically implanted human colon cancer cell line stably expressing a human sodium/iodide Symporter (NIS) reporter gene, which can be imaged with single-photon emission computed tomography (SPECT) using 99mTcO4−.
Jurgen Seidel - One of the best experts on this subject based on the ideXlab platform.
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ectopic expression of the sodium iodide Symporter enables imaging of transplanted cardiac stem cells in vivo by single photon emission computed tomography or positron emission tomography
Journal of the American College of Cardiology, 2008Co-Authors: John Terrovitis, Keng Fai Kwok, Riikka Lautamaki, James M Engles, Andreas S Barth, Eddy Kizana, Junichiro Miake, Michelle K Leppo, James Fox, Jurgen SeidelAbstract:Objectives: We examined the sodium-iodide Symporter (NIS), which promotes in vivo cellular uptake of technetium 99m (99mTc) or iodine 124 (124I), as a reporter gene for cell tracking by single-phot...
