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E Van Schaftingen - One of the best experts on this subject based on the ideXlab platform.
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Gene expression of glucokinase Regulatory Protein in regenerating rat liver
Hepatology, 1997Co-Authors: Jose Luis Rosa, E Van Schaftingen, Michel Detheux, J X Pérez, Ramon BartronsAbstract:The activity and messenger RNA (mRNA) levels of glucokinase, and the concentration and mRNA levels of its Regulatory Protein, were analyzed during liver regeneration. The activity of glucokinase and the concentration of its Regulatory Protein decreased to 30% and 50%, respectively, after liver resection, remaining low after 1 week. No significant variations in the level of these Proteins were found in sham-operated animals. The Regulatory Protein/glucokinase molar ratio increased during the replicative phase, to a maximum at 48 hours. The mRNA levels of glucokinase and of its Regulatory Protein decreased rapidly after partial hepatectomy to minimum values at 6 hours (15%) and at 12 hours (4%), respectively, returning to normal values at 24 hours and 168 hours, respectively. Sham-operated animals showed a similar decrease in mRNA levels during the prereplicative phase of liver regeneration, suggesting that the initial effects observed in the gene expression of these Proteins were due to surgical stress. During the replicative phase, a specific inhibition of the Regulatory Protein's gene expression was observed in the regenerating liver. A decrease in the content of Regulatory Protein and the glucokinase activity, and an increase in the molar ratio of these two Proteins correlate with the observed decrease in glycolytic flux, providing further evidence that the phosphorylation of glucose is a control point in the glycolytic/gluconeogenic flux during liver regeneration.
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Effect of mutations on the sensitivity of human beta-cell glucokinase to liver Regulatory Protein.
Diabetologia, 1996Co-Authors: Maria Veiga-da-cunha, Y. H. Lee, D. Marotta, Simon J. Pilkis, E Van SchaftingenAbstract:Human beta-cell glucokinase and its liver counterpart displayed a half-saturating concentration of glucose (S0.5) of about 8 mmol/l and a Hill coefficient of 1.7, and were as sensitive to inhibition by the rat liver Regulatory Protein as the rat liver enzyme. These results indicate that the N-terminal region of glucokinase, which differs among these three enzymes, is not implicated in the recognition of the Regulatory Protein. They also suggest that the Regulatory Protein, or a related Protein, could modulate the affinity of glucokinase for glucose in beta cells. We have also tested the effect of several mutations, many of which are implicated in maturity onset diabetes of the young. The mutations affected the affinity for glucose and for the Regulatory Protein to different degrees, indicating that the binding site for these molecules is different. An Asp158 Ala mutation, found in the expression plasmid previously thought to encode the wild-type enzyme, increased the affinity for glucose by about 2.5-fold without changing the affinity for the Regulatory Protein. The mutations that were found to decrease the affinity for the Regulatory Protein (Asn166 Arg, Val203 Ala, Asn204 Gln, Lys414 Ala) clustered in the hinge region of glucokinase and nearby in the large and small domains. These results are in agreement with the concept that part of the binding site for the Regulatory Protein is situated in the hinge region of this enzyme.
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the Regulatory Protein of glucokinase binds to the hepatocyte matrix but unlike glucokinase does not translocate during substrate stimulation
Biochemical Journal, 1995Co-Authors: Loranne Agius, Matthew Peak, E Van SchaftingenAbstract:The kinetic properties of hepatic glucokinase (hexokinase IV) are modulated by binding to a Regulatory Protein. This study shows that, in hepatocytes incubated with 5 mM glucose as sole carbohydrate substrate, both glucokinase and its Regulatory Protein bind to the cell matrix by a Mg(2+)-dependent mechanism. After incubation with an elevated [glucose] or with fructose, glucokinase, but not its Regulatory Protein, translocates from the Mg(2+)-dependent binding site. It is suggested that the Regulatory Protein acts as a receptor for anchoring glucokinase to the hepatocyte matrix and inhibiting its activity in metabolically quiescent conditions.
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Heterologous expression of an active rat Regulatory Protein of glucokinase
FEBS Letters, 1994Co-Authors: Michel Detheux, E Van SchaftingenAbstract:The cDNA presumed to encode the rat liver Regulatory Protein of glucokinase has been expressed in Escherichia coli and a partially soluble Protein has been obtained. This recombinant Protein was partially purified and found to have the same apparent molecular mass as the Regulatory Protein purified from rat liver. Like the latter, it inhibited rat liver glucokinase competitively with respect to glucose and its effect was sensitive to fructose 6-phosphate and fructose 1-phosphate.
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cloning and expression of a xenopus liver cdna encoding a fructose phosphate insensitive Regulatory Protein of glucokinase
FEBS Journal, 1994Co-Authors: Maria Veigadacunha, Michel Detheux, Nathalie Watelet, E Van SchaftingenAbstract:Xenopus liver contains a Protein inhibitor of glucokinase that, in contrast to the mammalian Regulatory Protein of glucokinase, is insensitive to fructose 6-phosphate and fructose 1-phosphate [Vandercammen A. & Van Schaftingen, E. (1993) Biochem. J. 294, 551-556]. The purpose of this work was to compare the primary structure and other properties of this Xenopus Protein with those of its rat liver counterpart. A Xenopus laevis liver cDNA library was screened using the cDNA encoding the rat liver Regulatory Protein as a probe. The cloned cDNA was 2534 bp long and encoded a 619-amino-acid Protein with a molecular mass of 68695 Da and 57% identity with the rat liver Regulatory Protein. This identity was only about 30% in an internal region (amino acids 349-381) and in the 70 carboxy terminal-residues. The Xenopus cDNA was expressed in Escherichia coli and the recombinant Regulatory Protein was purified to near homogeneity and found to have the same size, reactivity to antibodies and effects on the kinetics of glucokinase as the Protein purified from Xenopus liver. In contrast to the rat liver Regulatory Protein, both recombinant and native Xenopus Regulatory Proteins were insensitive to fructose 6-phosphate, fructose 1-phosphate and to physiological concentrations of Pi, and they inhibited Xenopus glucokinase with greater affinity than rat glucokinase. These results allow one to conclude that the fructose-phosphate-insensitive Protein of lower vertebrates is homologous to the fructose-6-phosphate-sensitive and fructose-1-phosphate-sensitive Protein found in mammals.
Michel Detheux - One of the best experts on this subject based on the ideXlab platform.
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Gene expression of glucokinase Regulatory Protein in regenerating rat liver
Hepatology, 1997Co-Authors: Jose Luis Rosa, E Van Schaftingen, Michel Detheux, J X Pérez, Ramon BartronsAbstract:The activity and messenger RNA (mRNA) levels of glucokinase, and the concentration and mRNA levels of its Regulatory Protein, were analyzed during liver regeneration. The activity of glucokinase and the concentration of its Regulatory Protein decreased to 30% and 50%, respectively, after liver resection, remaining low after 1 week. No significant variations in the level of these Proteins were found in sham-operated animals. The Regulatory Protein/glucokinase molar ratio increased during the replicative phase, to a maximum at 48 hours. The mRNA levels of glucokinase and of its Regulatory Protein decreased rapidly after partial hepatectomy to minimum values at 6 hours (15%) and at 12 hours (4%), respectively, returning to normal values at 24 hours and 168 hours, respectively. Sham-operated animals showed a similar decrease in mRNA levels during the prereplicative phase of liver regeneration, suggesting that the initial effects observed in the gene expression of these Proteins were due to surgical stress. During the replicative phase, a specific inhibition of the Regulatory Protein's gene expression was observed in the regenerating liver. A decrease in the content of Regulatory Protein and the glucokinase activity, and an increase in the molar ratio of these two Proteins correlate with the observed decrease in glycolytic flux, providing further evidence that the phosphorylation of glucose is a control point in the glycolytic/gluconeogenic flux during liver regeneration.
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Heterologous expression of an active rat Regulatory Protein of glucokinase
FEBS Letters, 1994Co-Authors: Michel Detheux, E Van SchaftingenAbstract:The cDNA presumed to encode the rat liver Regulatory Protein of glucokinase has been expressed in Escherichia coli and a partially soluble Protein has been obtained. This recombinant Protein was partially purified and found to have the same apparent molecular mass as the Regulatory Protein purified from rat liver. Like the latter, it inhibited rat liver glucokinase competitively with respect to glucose and its effect was sensitive to fructose 6-phosphate and fructose 1-phosphate.
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cloning and expression of a xenopus liver cdna encoding a fructose phosphate insensitive Regulatory Protein of glucokinase
FEBS Journal, 1994Co-Authors: Maria Veigadacunha, Michel Detheux, Nathalie Watelet, E Van SchaftingenAbstract:Xenopus liver contains a Protein inhibitor of glucokinase that, in contrast to the mammalian Regulatory Protein of glucokinase, is insensitive to fructose 6-phosphate and fructose 1-phosphate [Vandercammen A. & Van Schaftingen, E. (1993) Biochem. J. 294, 551-556]. The purpose of this work was to compare the primary structure and other properties of this Xenopus Protein with those of its rat liver counterpart. A Xenopus laevis liver cDNA library was screened using the cDNA encoding the rat liver Regulatory Protein as a probe. The cloned cDNA was 2534 bp long and encoded a 619-amino-acid Protein with a molecular mass of 68695 Da and 57% identity with the rat liver Regulatory Protein. This identity was only about 30% in an internal region (amino acids 349-381) and in the 70 carboxy terminal-residues. The Xenopus cDNA was expressed in Escherichia coli and the recombinant Regulatory Protein was purified to near homogeneity and found to have the same size, reactivity to antibodies and effects on the kinetics of glucokinase as the Protein purified from Xenopus liver. In contrast to the rat liver Regulatory Protein, both recombinant and native Xenopus Regulatory Proteins were insensitive to fructose 6-phosphate, fructose 1-phosphate and to physiological concentrations of Pi, and they inhibited Xenopus glucokinase with greater affinity than rat glucokinase. These results allow one to conclude that the fructose-phosphate-insensitive Protein of lower vertebrates is homologous to the fructose-6-phosphate-sensitive and fructose-1-phosphate-sensitive Protein found in mammals.
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short term control of glucokinase activity role of a Regulatory Protein
The FASEB Journal, 1994Co-Authors: E Van Schaftingen, Michel Detheux, Veiga M Da CunhaAbstract:Glucokinase is one of the four hexokinases present in mammalian tissues. It is expressed in two cell types that have to respond to changes in the blood glucose concentration, the liver parenchymal cell and the beta-cells of pancreatic islets. The former are responsible for the metabolism and storage of an important part of the ingested glucose, whereas the latter secrete insulin in response to an increase in the blood glucose level. One major characteristic of glucokinase is that it has a relatively low affinity for glucose and displays positive cooperativity for this substrate, despite the fact that it is a monomeric enzyme. Furthermore, unlike other hexokinases, it is not inhibited by micromolar (physiological) concentrations of glucose 6-phosphate but by a Regulatory Protein that transduces the effect of fructose 6-phosphate and of fructose 1-phosphate. The purpose of this review is to describe these aspects of the regulation of glucokinase.
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Cloning and sequencing of rat liver cDNAs encoding the Regulatory Protein of glucokinase.
FEBS Letters, 1993Co-Authors: Michel Detheux, J Vandekerckhove, E Van SchaftingenAbstract:cDNAs encoding the rat liver Regulatory Protein of glucokinase were cloned and sequenced. The deduced Protein contains 568 amino acids for a molecular mass of 62,867 Da. Northern blot analysis showed the presence of a major RNA species of 2.35 kb in rat liver. No signal was observed with muscle, brain, heart, testis, intestine or spleen RNA. Recombinant Regulatory Protein expressed in Escherichia coli was insoluble and inactive, and was presumably contained in inclusion bodies. Western blot analysis showed that the recombinant Protein was recognized by antibodies raised against Regulatory Protein purified from rat liver.
Loranne Agius - One of the best experts on this subject based on the ideXlab platform.
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Glucokinase Regulatory Protein is associated with mitochondria in hepatocytes
FEBS Letters, 2006Co-Authors: Catherine Arden, Simone Baltrusch, Loranne AgiusAbstract:Abstract The association of glucokinase with liver mitochondria has been reported [Danial et al. (2003) BAD and glucokinase reside in a mitochondrial complex that integrates glycolysis and apoptosis. Nature 424, 952–956]. We confirmed association of glucokinase immunoreactivity with rat liver mitochondria using Percoll gradient centrifugation and demonstrated its association with the 68 kDa Regulatory Protein (GKRP) but not with the binding Protein phosphofructokinase-2/fructose bisphosphatase-2. Substrates and glucagon induced adaptive changes in the mitochondrial glucokinase/GKRP ratio suggesting a Regulatory role for GKRP. Combined with previous observations that GKRP overexpression partially inhibits glycolysis [de la Iglesia et al. (2000) The role of the Regulatory Protein of glucokinase in the glucose sensory mechanism of the hepatocyte. J. Biol. Chem. 275, 10597–10603] these findings suggest that there may be distinct glycolytic pools of glucokinase.
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Evidence for glucose and sorbitol-induced nuclear export of glucokinase Regulatory Protein in hepatocytes
FEBS letters, 1999Co-Authors: Mohammed H. Mukhtar, Mark Stubbs, Loranne AgiusAbstract:Glucokinase is rapidly exported from the nucleus of hepatocytes in response to a rise in glucose or fructose 1-P. We demonstrate using confocal microscopy and quantitative imaging that in contrast to previous findings, the Regulatory Protein of glucokinase (GKRP) also translocates from the nucleus during substrate-induced translocation of glucokinase. However, the fractional decrease in nuclear GKRP is smaller than for glucokinase and is determined by the metabolic state and not by the distribution of glucokinase. Translocation of glucokinase and GKRP is not inhibited by leptomycin B, an inhibitor of exportin-1 function. These findings highlight the importance of quantitative imaging for determining nuclear export of Proteins and suggest that GKRP may have a role in nuclear export or import of glucokinase.
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the Regulatory Protein of glucokinase binds to the hepatocyte matrix but unlike glucokinase does not translocate during substrate stimulation
Biochemical Journal, 1995Co-Authors: Loranne Agius, Matthew Peak, E Van SchaftingenAbstract:The kinetic properties of hepatic glucokinase (hexokinase IV) are modulated by binding to a Regulatory Protein. This study shows that, in hepatocytes incubated with 5 mM glucose as sole carbohydrate substrate, both glucokinase and its Regulatory Protein bind to the cell matrix by a Mg(2+)-dependent mechanism. After incubation with an elevated [glucose] or with fructose, glucokinase, but not its Regulatory Protein, translocates from the Mg(2+)-dependent binding site. It is suggested that the Regulatory Protein acts as a receptor for anchoring glucokinase to the hepatocyte matrix and inhibiting its activity in metabolically quiescent conditions.
Tracey A. Rouault - One of the best experts on this subject based on the ideXlab platform.
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tempol mediated activation of latent iron Regulatory Protein activity prevents symptoms of neurodegenerative disease in irp2 knockout mice
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Manik C Ghosh, Winghang Tong, Deliang Zhang, Hayden Ollivierrewilson, Anamika Singh, Murali C Krishna, James B Mitchell, Tracey A. RouaultAbstract:In mammals, two homologous cytosolic Regulatory Proteins, iron Regulatory Protein 1 (also known as IRP1 and Aco1) and iron Regulatory Protein 2 (also known as IRP2 and Ireb2), sense cytosolic iron levels and posttranscriptionally regulate iron metabolism genes, including transferrin receptor 1 (TfR1) and ferritin H and L subunits, by binding to iron-responsive elements (IREs) within target transcripts. Mice that lack IRP2 develop microcytic anemia and neurodegeneration associated with functional cellular iron depletion caused by low TfR1 and high ferritin expression. IRP1 knockout (IRP1−/−) animals do not significantly misregulate iron metabolism, partly because IRP1 is an iron-sulfur Protein that functions mainly as a cytosolic aconitase in mammalian tissues and IRP2 activity increases to compensate for loss of the IRE binding form of IRP1. The neurodegenerative disease of IRP2−/− animals progresses slowly as the animals age. In this study, we fed IRP2−/− mice a diet supplemented with a stable nitroxide, Tempol, and showed that the progression of neuromuscular impairment was markedly attenuated. In cell lines derived from IRP2−/− animals, and in the cerebellum, brainstem, and forebrain of animals maintained on the Tempol diet, IRP1 was converted from a cytosolic aconitase to an IRE binding Protein that stabilized the TfR1 transcript and repressed ferritin synthesis. We suggest that Tempol protected IRP2−/− mice by disassembling the cytosolic iron-sulfur cluster of IRP1 and activating IRE binding activity, which stabilized the TfR1 transcript, repressed ferritin synthesis, and partially restored normal cellular iron homeostasis in the brain.
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iron Regulatory Protein 2 as iron sensor iron dependent oxidative modification of cysteine
Journal of Biological Chemistry, 2003Co-Authors: Daekyung Kang, Tracey A. Rouault, Steven K Drake, Nancy B Wehr, Jinsook Jeong, Rodney L. LevineAbstract:Iron Regulatory Protein 2 coordinates cellular regulation of iron metabolism by binding to iron responsive elements in mRNA. The Protein is synthesized constitutively but is rapidly degraded when iron stores are replete. This iron-dependent degradation requires the presence of a 73-residue degradation domain, but its functions have not yet been established. We now show that the domain can act as an iron sensor, mediating its own covalent modification. The domain forms an iron-binding site with three cysteine residues located in the middle of the domain. It then reacts with molecular oxygen to generate a reactive oxidizing species at the iron-binding site. One cysteine residue is oxidized to dehydrocysteine and other products. This covalent modification may thus mark the Protein molecule for degradation by the proteasome system.
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iron dependent oxidation ubiquitination and degradation of iron Regulatory Protein 2 implications for degradation of oxidized Proteins
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Kazuhiro Iwai, Rodney L. Levine, Steven K Drake, Nancy B Wehr, Allan M Weissman, Timothy Lavaute, Nagahiro Minato, Richard D Klausner, Tracey A. RouaultAbstract:The ability of iron to catalyze formation of reactive oxygen species significantly contributes to its toxicity in cells and animals. Iron uptake and distribution is regulated tightly in mammalian cells, in part by iron Regulatory Protein 2 (IRP2), a Protein that is degraded efficiently by the proteasome in iron-replete cells. Here, we demonstrate that IRP2 is oxidized and ubiquitinated in cells before degradation. Moreover, iron-dependent oxidation converts IRP2 into a substrate for ubiquitination in vitro. A Regulatory pathway is described in which excess iron is sensed by its ability to catalyze site-specific oxidations in IRP2, oxidized IRP2 is ubiquitinated, and ubiquitinated IRP2 subsequently is degraded by the proteasome. Selective targeting and removal of oxidatively modified Proteins may contribute to the turnover of many Proteins that are degraded by the proteasome.
Matthias W Hentze - One of the best experts on this subject based on the ideXlab platform.
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iron Regulatory Protein 1 sustains mitochondrial iron loading and function in frataxin deficiency
Cell Metabolism, 2015Co-Authors: Alain Martelli, Matthias W Hentze, Bruno Galy, Stephane Schmucker, Laurence Reutenauer, Jacques Mathieu, Carole Peyssonnaux, Zoubida Karim, Herve Puy, Helene PuccioAbstract:Mitochondrial iron accumulation is a hallmark of diseases associated with impaired iron-sulfur cluster (Fe-S) biogenesis, such as Friedreich ataxia linked to frataxin (FXN) deficiency. The pathophysiological relevance of the mitochondrial iron loading and the underlying mechanisms are unknown. Using a mouse model of hepatic FXN deficiency in combination with mice deficient for iron Regulatory Protein 1 (IRP1), a key regulator of cellular iron metabolism, we show that IRP1 activation in conditions of Fe-S deficiency increases the available cytosolic labile iron pool. Surprisingly, our data indicate that IRP1 activation sustains mitochondrial iron supply and function rather than driving detrimental iron overload. Mitochondrial iron accumulation is shown to depend on mitochondrial dysfunction and heme-dependent upregulation of the mitochondrial iron importer mitoferrin-2. Our results uncover an unexpected protective role of IRP1 in pathological conditions associated with altered Fe-S metabolism.
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systemic iron homeostasis and the iron responsive element iron Regulatory Protein ire irp Regulatory network
Annual Review of Nutrition, 2008Co-Authors: Martina U Muckenthaler, Bruno Galy, Matthias W HentzeAbstract:The regulation and maintenance of systemic iron homeostasis is critical to human health. Iron overload and deficiency diseases belong to the most common nutrition-related pathologies across the globe. It is now well appreciated that the hormonal hepcidin/ferroportin system plays an important Regulatory role for systemic iron metabolism. We review recent data that uncover the importance of the cellular iron-responsive element/iron-Regulatory Protein (IRE/IRP) Regulatory network in systemic iron homeostasis. We also discuss how the IRE/IRP Regulatory system communicates with the hepcidin/ferroportin system to connect the control networks for systemic and cellular iron balance.
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iron Regulatory Protein prevents binding of the 43s translation pre initiation complex to ferritin and ealas mrnas
The EMBO Journal, 1994Co-Authors: Nicola K Gray, Matthias W HentzeAbstract:Translation of ferritin and erythroid 5-aminolevulinate synthase (eALAS) mRNAs is regulated by iron via mRNA-Protein interactions between iron-responsive elements (IREs) and iron Regulatory Protein (IRP). In iron-depleted cells, IRP binds to single IREs located in the 5' untranslated regions of ferritin and eALAS mRNAs and represses translation initiation. The molecular mechanism underlying this translational repression was investigated using reconstituted, IRE-IRP-regulated, cell-free translation systems. The IRE-IRP interaction is shown to prevent the association of the 43S translation pre-initiation complex (including the small ribosomal subunit) with the mRNA. Studies with the spliceosomal Protein U1A and mRNAs which harbour specific binding sites for this Protein in place of an IRE furthermore reveal that the 5' termini of mRNAs are generally sensitive to repressor Protein-mediated inhibition of 43S pre-initiation complex binding.
