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Sophie Lemoine - One of the best experts on this subject based on the ideXlab platform.
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physiological and toxic effects of purine intermediate 5 amino 4 Imidazolecarboxamide ribonucleotide aicar in yeast
Journal of Biological Chemistry, 2011Co-Authors: Hans Caspar Hurlimann, Benoit Laloo, Barbara Simonkayser, Christelle Saintmarc, Fanny Coulpier, Sophie LemoineAbstract:5-Amino-4-Imidazolecarboxamide ribonucleotide 5′-phosphate (AICAR) is a monophosphate metabolic intermediate of the de novo purine synthesis pathway that has highly promising metabolic and antiproliferative properties. Yeast mutants unable to metabolize AICAR are auxotroph for histidine. A screening for suppressors of this phenotype identified recessive and dominant mutants that result in lowering the intracellular AICAR concentration. The recessive mutants affect the adenosine kinase, which is shown here to catalyze the phosphorylation of AICAR riboside in yeast. The dominant mutants strongly enhance the capacity of the alkaline phosphatase Pho13 to dephosphorylate 5-amino-4-imidazole N-succinocarboxamide ribonucleotide 5′-phosphate(SAICAR) into its non-toxic riboside form. By combining these mutants with transcriptomics and metabolomics analyses, we establish that in yeast responses to AICAR and SAICAR are clearly linked to the concentration of the monophosphate forms, whereas the derived nucleoside moieties have no effect even at high intracellular concentration. Finally, we show that AICAR/SAICAR concentrations vary under physiological conditions known to modulate transcription of the purine and phosphate pathway genes.
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physiological and toxic effects of purine intermediate 5 amino 4 Imidazolecarboxamide ribonucleotide
2011Co-Authors: Benoit Laloo, Barbara Simonkayser, Christelle Saintmarc, Fanny Coulpier, Sophie Lemoine, Bertrand Daignanfornier, Benoit PinsonAbstract:5-Amino-4-Imidazolecarboxamide ribonucleotide 5-phosphate (AICAR) is a monophosphate metabolic intermediate of the de novo purine synthesis pathway that has highly promising metabolic and antiproliferative properties. Yeast mutants unable to metabolize AICAR are auxotroph for histidine. A screening for suppressors of this phenotype identified recessive and dominant mutants that result in lowering the intracellular AICAR concentration. The recessive mutants affect the adenosine kinase, which is shown here to catalyze the phosphorylation of AICAR riboside in yeast. The dominant mutants strongly enhance the capacity of the alkaline phosphatase Pho13 to dephosphorylate 5-amino-4-imidazole N-succinocarboxamide ribonucleotide 5-phosphate(SAICAR) into its non-toxic riboside form. By combining these mutants with transcriptomics and metabolomics analyses, we establish that in yeast responses to AICAR and SAICAR are clearly linked to the concentration of the monophosphate forms, whereas the derived nucleoside moieties have no effect even at high intracellular concentration. Finally, we show that AICAR/SAICAR concentrations vary under physiological conditions known to modulate transcription of the purine and phosphate pathway genes.
Karen S Anderson - One of the best experts on this subject based on the ideXlab platform.
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the kinetic mechanism of the human bifunctional enzyme atic 5 amino 4 Imidazolecarboxamide ribonucleotide transformylase inosine 5 monophosphate cyclohydrolase a surprising lack of substrate channeling
Journal of Biological Chemistry, 2002Co-Authors: Karen G Bulock, Peter G Beardsley, Karen S AndersonAbstract:Abstract 5-Amino-4-Imidazolecarboxamide ribonucleotide transformylase/IMP cyclohydrolase (ATIC) is a bifunctional protein possessing two enzymatic activities that sequentially catalyze the last two steps in the pathway forde novo synthesis of inosine 5′-monophosphate. This bifunctional enzyme is of particular interest because of its potential as a chemotherapeutic target. Furthermore, these two catalytic activities reside on the same protein throughout all of nature, raising the question of whether there is some kinetic advantage to the bifunctionality. Rapid chemical quench, stopped-flow absorbance, and steady-state kinetic techniques were used to elucidate the complete kinetic mechanism of human ATIC. The kinetic simulation program KINSIM was used to model the kinetic data obtained in this study. The detailed kinetic analysis, in combination with kinetic simulations, provided the following key features of the enzyme reaction pathway. 1) The rate-limiting step in the overall reaction (2.9 ± 0.4 s−1) is likely the release of tetrahydrofolate from the formyltransferase active site or a conformational change associated with tetrahydrofolate release. 2) The rate of the reverse transformylase reaction (6.7 s−1) is ∼2–3-fold faster than the forward rate (2.9 s−1), whereas the cyclohydrolase reaction is essentially unidirectional in the forward sense. The cyclohydrolase reaction thus draws the overall bifunctional reaction toward the production of inosine monophosphate. 3) There was no kinetic evidence of substrate channeling of the intermediate, the formylaminoimidazole carboxamide ribonucleotide, between the formyltransferase and the cyclohydrolase active sites.
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the kinetic mechanism of the human bifunctional enzyme atic 5 amino 4 Imidazolecarboxamide ribonucleotide transformylase inosine 5 monophosphate cyclohydrolase a surprising lack of substrate channeling
Journal of Biological Chemistry, 2002Co-Authors: Karen G Bulock, Peter G Beardsley, Karen S AndersonAbstract:5-Amino-4-Imidazolecarboxamide ribonucleotide transformylase/IMP cyclohydrolase (ATIC) is a bifunctional protein possessing two enzymatic activities that sequentially catalyze the last two steps in the pathway for de novo synthesis of inosine 5'-monophosphate. This bifunctional enzyme is of particular interest because of its potential as a chemotherapeutic target. Furthermore, these two catalytic activities reside on the same protein throughout all of nature, raising the question of whether there is some kinetic advantage to the bifunctionality. Rapid chemical quench, stopped-flow absorbance, and steady-state kinetic techniques were used to elucidate the complete kinetic mechanism of human ATIC. The kinetic simulation program KINSIM was used to model the kinetic data obtained in this study. The detailed kinetic analysis, in combination with kinetic simulations, provided the following key features of the enzyme reaction pathway. 1) The rate-limiting step in the overall reaction (2.9 +/- 0.4 s(-1)) is likely the release of tetrahydrofolate from the formyltransferase active site or a conformational change associated with tetrahydrofolate release. 2) The rate of the reverse transformylase reaction (6.7 s(-1)) is approximately 2-3-fold faster than the forward rate (2.9 s(-1)), whereas the cyclohydrolase reaction is essentially unidirectional in the forward sense. The cyclohydrolase reaction thus draws the overall bifunctional reaction toward the production of inosine monophosphate. 3) There was no kinetic evidence of substrate channeling of the intermediate, the formylaminoimidazole carboxamide ribonucleotide, between the formyltransferase and the cyclohydrolase active sites.
Fanny Coulpier - One of the best experts on this subject based on the ideXlab platform.
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physiological and toxic effects of purine intermediate 5 amino 4 Imidazolecarboxamide ribonucleotide aicar in yeast
Journal of Biological Chemistry, 2011Co-Authors: Hans Caspar Hurlimann, Benoit Laloo, Barbara Simonkayser, Christelle Saintmarc, Fanny Coulpier, Sophie LemoineAbstract:5-Amino-4-Imidazolecarboxamide ribonucleotide 5′-phosphate (AICAR) is a monophosphate metabolic intermediate of the de novo purine synthesis pathway that has highly promising metabolic and antiproliferative properties. Yeast mutants unable to metabolize AICAR are auxotroph for histidine. A screening for suppressors of this phenotype identified recessive and dominant mutants that result in lowering the intracellular AICAR concentration. The recessive mutants affect the adenosine kinase, which is shown here to catalyze the phosphorylation of AICAR riboside in yeast. The dominant mutants strongly enhance the capacity of the alkaline phosphatase Pho13 to dephosphorylate 5-amino-4-imidazole N-succinocarboxamide ribonucleotide 5′-phosphate(SAICAR) into its non-toxic riboside form. By combining these mutants with transcriptomics and metabolomics analyses, we establish that in yeast responses to AICAR and SAICAR are clearly linked to the concentration of the monophosphate forms, whereas the derived nucleoside moieties have no effect even at high intracellular concentration. Finally, we show that AICAR/SAICAR concentrations vary under physiological conditions known to modulate transcription of the purine and phosphate pathway genes.
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physiological and toxic effects of purine intermediate 5 amino 4 Imidazolecarboxamide ribonucleotide
2011Co-Authors: Benoit Laloo, Barbara Simonkayser, Christelle Saintmarc, Fanny Coulpier, Sophie Lemoine, Bertrand Daignanfornier, Benoit PinsonAbstract:5-Amino-4-Imidazolecarboxamide ribonucleotide 5-phosphate (AICAR) is a monophosphate metabolic intermediate of the de novo purine synthesis pathway that has highly promising metabolic and antiproliferative properties. Yeast mutants unable to metabolize AICAR are auxotroph for histidine. A screening for suppressors of this phenotype identified recessive and dominant mutants that result in lowering the intracellular AICAR concentration. The recessive mutants affect the adenosine kinase, which is shown here to catalyze the phosphorylation of AICAR riboside in yeast. The dominant mutants strongly enhance the capacity of the alkaline phosphatase Pho13 to dephosphorylate 5-amino-4-imidazole N-succinocarboxamide ribonucleotide 5-phosphate(SAICAR) into its non-toxic riboside form. By combining these mutants with transcriptomics and metabolomics analyses, we establish that in yeast responses to AICAR and SAICAR are clearly linked to the concentration of the monophosphate forms, whereas the derived nucleoside moieties have no effect even at high intracellular concentration. Finally, we show that AICAR/SAICAR concentrations vary under physiological conditions known to modulate transcription of the purine and phosphate pathway genes.
Marie-françoise Vincent - One of the best experts on this subject based on the ideXlab platform.
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substrate cycling between 5 amino 4 Imidazolecarboxamide riboside and its monophosphate in isolated rat hepatocytes
Biochemical Pharmacology, 1996Co-Authors: Marie-françoise Vincent, Francoise Bontemps, Georges Van Den BergheAbstract:AICA (5-amino-4-Imidazolecarboxamide)-riboside is taken up by isolated rat hepatocytes and converted by adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) into AICAR (ZMP), an intermediate of the de novo synthesis of purine nucleotides. We investigated if, in these cells, a cycle analogous to the adenosine-AMP substrate cycle operates between AICAriboside and ZMP. When 50 microM ITu, an inhibitor of adenosine kinase, was added to hepatocytes that had metabolized AICAriboside for 30 min, the concentration of ZMP decreased immediately. This was mirrored by a reincrease of AICAriboside. Rates of the ITu-induced decrease of ZMP and the increase of AICAriboside, calculated at different concentrations of ZMP, were first order, up to the highest concentration of ZMP (approx. 5 mumol/g of cells). Dephosphorylation of ZMP added to crude cytosolic extracts of rat liver displayed hyperbolic kinetics, with a Vmax of 0.65 mumol/min per g protein and an apparent Km of 5 mM, and was markedly inhibited by Pi, an inhibitor of IMP-GMP 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5). We conclude that hepatocyte ZMP is continuously dephosphorylated, most likely by IMP-GMP 5'-nucleotidase, into AICAriboside, which is rephosphorylated into ZMP by adenosine kinase. Substrate cycling was also shown to occur between other nucleoside analogs and their phosphorylated counterparts.
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Inhibition of fatty acid and cholesterol synthesis by stimulation of AMP-activated protein kinase.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1995Co-Authors: Nathalie Henin, Marie-françoise Vincent, H E Gruber, G Van Den BergheAbstract:AMP-activated protein kinase is a multisubstrate protein kinase that, in liver, inactivates both acetyl-CoA carboxylase, the rate-limiting enzyme of fatty acid synthesis, and 3-hydroxy-3-methyl-glutaryl-CoA reductase, the rate-limiting enzyme of cholesterol synthesis. AICAR (5-amino 4-Imidazolecarboxamide ribotide, ZMP) was found to stimulate up to 10-fold rat liver AMP-activated protein kinase, with a half-maximal effect at approximately 5 mM. In accordance with previous observations, addition to suspensions of isolated rat hepatocytes of 50-500 microM AICAriboside, the nucleoside corresponding to ZMP, resulted in the accumulation of millimolar concentrations of the latter. This was accompanied by a dose-dependent inactivation of both acetyl-CoA carboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase. Addition of 50-500 microM AICAriboside to hepatocyte suspensions incubated in the presence of various substrates, including glucose and lactate/pyruvate, caused a parallel inhibition of both fatty acid and cholesterol synthesis. With lactate/pyruvate (10/1 mM), half-maximal inhibition was obtained at approximately 100 microM, and near-complete inhibition at 500 microM AICAriboside. These findings open new perspectives for the simultaneous control of triglyceride and cholesterol synthesis by pharmacological stimulators of AMP-activated protein kinase.
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inhibition of glycolysis by 5 amino 4 Imidazolecarboxamide riboside in isolated rat hepatocytes
Biochemical Journal, 1992Co-Authors: Marie-françoise Vincent, Francoise Bontemps, G Van Den BergheAbstract:5-Amino-4-Imidazolecarboxamide riboside (AICAriboside; Z-riboside), the nucleotide corresponding to AICAribotide (AICAR or ZMP), an intermediate of the 'de novo' pathway of purine nucleotide biosynthesis, has been shown to inhibit gluconeogenesis in isolated rat hepatocytes [Vincent, Marangos, Gruber & Van den Berghe (1991) Diabetes 40, 1259-1266]. We now report that glycosis is also inhibited and even more sensitive to AICAriboside in these cells. In hepatocyte suspensions from fasted rats, production of lactate from 15 mM-glucose was half-maximally inhibited by 25-50 microM-AICAriboside. AICAriboside influenced two regulatory steps of glycolysis: (1) it decreased the release of 3H2O from [2-3H]glucose and the concentrations of both glucose 6-phosphate and fructose 6-phosphate, indicating that it diminished the phosphorylation of glucose by glucokinase; (2) it decreased the concentration of fructose 2,6-bisphosphate (Fru-2,6-P2), the main physiological stimulator of liver 6-phosphofructo-1-kinase. Further studies showed that AICAriboside induced an inactivation of 6-phosphofructo-2-kinase, the enzyme that produces Fru-2,6-P2, without affecting the concentration of cyclic AMP. Similarly to the inhibiton of gluconeogenesis by AICAriboside, the inhibition of glycolysis became apparent after an approx. 10 min latency and persisted when the cells were washed after addition of AICAriboside, strongly suggesting that the effects were also exerted by the Z-nucleotides, which accumulate after addition of AICAriboside to hepatocytes. An increased uptake of lactate was evident when 50-200 microM-AICAriboside was added 15 min after addition of glucose. This can be explained by the higher sensitivity of glycolysis, as compared with gluconeogenesis, to inhibition by AICAriboside, and reveals the simultaneous operation of both processes.
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Inhibition by AICA riboside of gluconeogenesis in isolated rat hepatocytes.
Diabetes, 1991Co-Authors: Marie-françoise Vincent, Paul J. Marangos, Harry E. Gruber, G Van Den BergheAbstract:5-Amino-4-Imidazolecarboxamide (AICA) riboside, the nucleoside corresponding to AICA ribotide (AICAR or ZMP), an intermediate of the de novo pathway of purine biosynthesis, was found to exert a dose-dependent inhibition on gluconeogenesis in isolated rat hepatocytes. Production of glucose from lactate-pyruvate mixtures was half-maximally inhibited by approximately 100 microM and completely suppressed by 500 microM AICA riboside. AICA riboside also inhibited the production of glucose from all other gluconeogenic precursors investigated, i.e., fructose, dihydroxyacetone, and L-proline. Measurements of intermediates of the glycolytic-gluconeogenic pathway showed that AICA riboside provoked elevations of triose phosphates and fructose-1,6-bisphosphate and decreases in fructose-6-phosphate and glucose-6-phosphate. The effects of AICA riboside persisted when the cells were washed 10 min after its addition but were suppressed by 5-iodotubercidin, an inhibitor of adenosine kinase. AICA riboside provoked a dose-dependent buildup of normally undetectable Z nucleotides. After 20 min of incubation with 500 microM AICA riboside, ZMP, ZTP, and ZDP reached 3, 0.3, and 0.1 mumol/g cells, respectively. Concentrations of ATP were not significantly modified by addition of up to 500 microM AICA riboside when the cells were incubated with lactate-pyruvate but decreased with fructose or dihydroxyacetone. The activity of rat liver fructose-1,6-bisphosphatase was inhibited by ZMP with an apparent Ki of 370 microM. It is concluded that AICA riboside exerts a suppressive effect on gluconeogenesis because it provokes an accumulation of ZMP, which inhibits fructose-1,6-bisphosphatase.(ABSTRACT TRUNCATED AT 250 WORDS)
Franck Dequiedt - One of the best experts on this subject based on the ideXlab platform.
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Phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) is an AMPK target participating in contraction-stimulated glucose uptake in skeletal muscle.
The Biochemical journal, 2013Co-Authors: Yang Liu, Yu-chiang Lai, Elaine V. Hill, Donatienne Tyteca, Sarah Carpentier, Ada Ingvaldsen, Didier Vertommen, Louise Lantier, Marc Foretz, Franck DequiedtAbstract:PIKfyve (FYVE domain-containing phosphatidylinositol 3-phosphate 5-kinase), the lipid kinase that phosphorylates PtdIns3P to PtdIns(3,5)P2, has been implicated in insulin-stimulated glucose uptake. We investigated whether PIKfyve could also be involved in contraction/AMPK (AMP-activated protein kinase)-stimulated glucose uptake in skeletal muscle. Incubation of rat epitrochlearis muscles with YM201636, a selective PIKfyve inhibitor, reduced contraction- and AICAriboside (5-amino-4-Imidazolecarboxamide riboside)-stimulated glucose uptake. Consistently, PIKfyve knockdown in C2C12 myotubes reduced AICAriboside-stimulated glucose transport. Furthermore, muscle contraction increased PtdIns(3,5)P2 levels and PIKfyve phosphorylation. AMPK phosphorylated PIKfyve at Ser307 both in vitro and in intact cells. Following subcellular fractionation, PIKfyve recovery in a crude intracellular membrane fraction was increased in contracting versus resting muscles. Also in opossum kidney cells, wild-type, but not S307A mutant, PIKfyve was recruited to endosomal vesicles in response to AMPK activation. We propose that PIKfyve activity is required for the stimulation of skeletal muscle glucose uptake by contraction/AMPK activation. PIKfyve is a new AMPK substrate whose phosphorylation at Ser307 could promote PIKfyve translocation to endosomes for PtdIns(3,5)P2 synthesis to facilitate GLUT4 (glucose transporter 4) translocation.
