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Jacques Ricard - One of the best experts on this subject based on the ideXlab platform.
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information transfer in multienzyme complexes 1 thermodynamics of conformational constraints and memory effects in the bienzyme Glyceraldehyde 3 phosphate dehydrogenase phosphoribulokinase complex of chlamydomonas reinhardtii chloroplasts
FEBS Journal, 1997Co-Authors: Sandrine Lebreton, Brigitte Gontero, Luisana Avilan, Jacques RicardAbstract:Oxidized phosphoribulokinase is almost inactive in its isolated state but becomes active when associated with Glyceraldehyde-3-phosphate dehydrogenase. There is therefore an information transfer that takes place between these two enzymes. However, when the complex dissociates, free oxidized phosphoribulokinase is even more active than when it is associated with Glyceraldehyde-3-phosphate dehydrogenase. This means that Glyceraldehyde-3-phosphate dehydrogenase exerts an imprinting effect upon phosphoribulokinase which persists for a while after the parting of the two proteins. Various methods derived from statistical thermodynamics can be used to estimate the fraction of energy transferred from Glyceraldehyde-3-phosphate dehydrogenase to phosphoribulokinase and which alters the kinetic parameters of the latter enzyme. In the complex, the decrease of the free energy associated with the binding of ribulose 5-phosphate is larger than that of ATP. This implies that the mutual association of the two enzymes facilitates the binding of the former substrate but is without effect on that of the latter. The main effect exerted by the association of the two enzymes is to decrease by about 10 kJ/ mol the height of the energy barrier of the catalytic process. Phosphoribulokinase keeps an imprinting effect exerted by Glyceraldehyde-3-phosphate dehydrogenase after the parting of the two enzymes. Part of the energy transferred from one protein to the other is used to decrease slightly the apparent binding free energy of the two substrates of phosphoribulokinase by about 1.5 kJ/mol. Whereas the previous association of the two enzymes does not significantly alter substrate binding to phosphoribulokinase, it greatly affects catalysis and decreases by about 16 kJ/mol the height of the energy barrier pertaining to this step. Therefore, within multienzyme complexes, information and energy can be transferred between proteins. Statistical thermodynamics offers the possibility of estimating how this energy is used to alter the various kinetic parameters of the reaction.
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memory and imprinting effects in multienzyme complexes i isolation dissociation and reassociation of a phosphoribulokinase Glyceraldehyde 3 phosphate dehydrogenase complex from chlamydomonas reinhardtii chloroplasts
FEBS Journal, 1997Co-Authors: Luisana Avilan, Sandrine Lebreton, Brigitte Gontero, Jacques RicardAbstract:A bienzyme complex made up of phosphoribulokinase and Glyceraldehyde-3-phosphate dehydrogenase has been isolated and purified from chloroplasts of Chlamydomonas reinhardtii. The complex contains four phosphoribulokinase and eight Glyceraldehyde-3-phosphate dehydrogenase polypeptide chains. As phosphoribulokinase is dimeric and Glyceraldehyde-3-phosphate dehydrogenase tetrameric, it is concluded that the complex comprises two phosphoribulokinase and two Glyceraldehyde-3-phosphate dehydrogenase molecules. Its overall molecular mass is 460 kDa, which is in excellent agreement with its stoichiometry. Moreover, owing to the nature of the two enzymes, this complex must catalyse two nonconsecutive reactions. The bienzyme complex tended to spontaneously dissociate into the free enzymes upon dilution. This dissociation process was considerably promoted by reducing agents such as dithiothreitol or reduced thioredoxin. The kinetics of the dissociation process induced by dithiothreitol or reduced thioredoxin were paralleled by an increase of activity of phosphoribulokinase. The dissociation of the complex was reversible. If oxidized phosphoribulokinase and Glyceraldehyde-3-phosphate dehydrogenase were mixed, a certain amount of the complex was formed. The reconstituted complex displayed properties that were indistinguishable from those of the native complex extracted from chloroplasts of Chlamydomonas reinhardtii. These results suggest that the concentration of the complex in vivo must vary depending on the light intensity.
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memory and imprinting effects in multienzyme complexes ii kinetics of the bienzyme complex from chlamydomonas reinhardtii and hysteretic activation of chloroplast oxidized phosphoribulokinase
FEBS Journal, 1997Co-Authors: Sandrine Lebreton, Brigitte Gontero, Luisana Avilan, Jacques RicardAbstract:Oxidized, free, stable phosphoribulokinase from Chlamydomonas reinhardtii was almost completely devoid of catalytic activity (0.06 s(-1)/site). However, when it was bound to Glyceraldehyde-3-phosphate dehydrogenase from the same organism, it displayed significant activity (3.25 s(-1)/site). Moreover, this complex tended to spontaneously dissociate upon dilution; the isolated phosphoribulokinase activity increased up to 56 s(-1)/site, subsequently decreased, and finally became almost completely inactive. Its intrinsic kinetic properties (Km and k(cat)) changed with the variation of the overall activity. These effects were paralleled by changes of conformation of the enzyme as revealed by fluorescence analysis. A model is proposed that allows quantitative expression of the dynamics of the dissociation of the oxidized bienzyme complex and the effects of either of the two substrates, ATP and ribulose 5-phosphate, on this dissociation process. Whereas ATP destabilized the complex and promoted its dissociation, ribulose 5-phosphate tended to stabilize this complex. Inactive, stable, oxidized phosphoribulokinase may form a complex with Glyceraldehyde-3-phosphate dehydrogenase regaining its catalytic activity. In this case, Glyceraldehyde-3-phosphate dehydrogenase acts in a manner similar, but not identical to a chaperonin. The information content of the phosphoribulokinase gene, as defined by the sequence of its base pairs, was therefore not sufficient to specify full enzyme activity. It needed the presence of Glyceraldehyde-3-phosphate dehydrogenase to give the oxidized phosphoribulokinase a conformation competent for its activity. The potential biological significance of these effects remains to be discovered.
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structural and functional properties of a multi enzyme complex from spinach chloroplasts 2 modulation of the kinetic properties of enzymes in the aggregated state
FEBS Journal, 1993Co-Authors: Brigitte Gontero, Guillermo Mulliert, Magali Rault, Marietherese Giudiciorticoni, Jacques RicardAbstract:Antibodies have been raised specifically against chloroplast phosphoribulokinase, Glyceraldehyde-3-phosphate dehydrogenase and ribulose 1,5-bisphosphate carboxylase-oxygenase. Each of these antibodies recognizes the same macromolecular entity isolated and purified from chloroplasts. This entity is a multi-enzyme complex, previously isolated and made up of ribose-phosphate isomerase, phosphoribulokinase, ribulose 1,5-bisphosphate carboxylase-oxygenase, phosphoglycerate kinase and Glyceraldehyde-3-phosphate dehydrogenase. Under denaturing conditions the multi-enzyme complex contains two polypeptides of 54 kDa and 15 kDa corresponding to the large and the small subunits of ribulose 1,5-bisphosphate carboxylase-oxygenase, the two polypeptides of the Glyceraldehyde-3-phosphate dehydrogenase of 39 kDa and 37 kDa, one polypeptide of 40 kDa pertaining to phosphoribulokinase and one polypeptide of 30 kDa very likely pertaining to ribose-phosphate isomerase. The combined use of immunochemical and densitometric techniques allows one to determine the number and the stoichiometry of the various types of polypeptide chains and to compare them with the quaternary structure of the corresponding isolated enzymes. Ribulose 1,5-bisphosphate carboxylase-oxygenase of higher plants consists of eight large and eight small subunits. Glyceraldehyde-3-phosphate dehydrogenase is made up of two types of polypeptide chains called A and B and its simplest quaternary structure is A2B2. Finally, phosphoribulokinase is a dimer made up of two identical subunits. Therefore, for the three isolated enzymes, the stoichiometry of the polypeptide chains is always 1:1. Within this multi-enzyme complex, there are two subunits of phosphoribulokinase, two A and B subunits of Glyceraldehyde-3-phosphate dehydrogenase and two large and four small subunits of ribulose 1,5-bisphosphate carboxylase-oxygenase. Therefore the number and the stoichiometry of the polypeptide chains of phosphoribulokinase and Glyceraldehyde-3-phosphate dehydrogenase are the same in the multi-enzyme complex and in the free enzymes, but those of ribulose 1,5-bisphosphate carboxylase-oxygenase are completely different. This conclusion that the multi-enzyme complex contains two active sites for ribulose 1,5-bisphosphate may be confirmed independently by kinetic inhibition studies using 6-phosphogluconate.
James Ronald Gillig - One of the best experts on this subject based on the ideXlab platform.
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pharmacological inhibition of nicotinamide phosphoribosyltransferase nampt an enzyme essential for nad biosynthesis in human cancer cells metabolic basis and potential clinical implications
Journal of Biological Chemistry, 2013Co-Authors: Bo Tan, Robert L Shepard, K Roth, Debra A Young, Tao Wang, Timothy I Meier, Yan Zhai, Karen L Huss, Mingshang Kuo, James Ronald GilligAbstract:Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the first rate-limiting step in converting nicotinamide to NAD+, essential for cellular metabolism, energy production, and DNA repair. NAMPT has been extensively studied because of its critical role in these cellular processes and the prospect of developing therapeutics against the target, yet how it regulates cellular metabolism is not fully understood. In this study we utilized liquid chromatography-mass spectrometry to examine the effects of FK866, a small molecule inhibitor of NAMPT currently in clinical trials, on glycolysis, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, and serine biosynthesis in cancer cells and tumor xenografts. We show for the first time that NAMPT inhibition leads to the attenuation of glycolysis at the Glyceraldehyde 3-phosphate dehydrogenase step due to the reduced availability of NAD+ for the enzyme. The attenuation of glycolysis results in the accumulation of glycolytic intermediates before and at the Glyceraldehyde 3-phosphate dehydrogenase step, promoting carbon overflow into the pentose phosphate pathway as evidenced by the increased intermediate levels. The attenuation of glycolysis also causes decreased glycolytic intermediates after the Glyceraldehyde 3-phosphate dehydrogenase step, thereby reducing carbon flow into serine biosynthesis and the TCA cycle. Labeling studies establish that the carbon overflow into the pentose phosphate pathway is mainly through its non-oxidative branch. Together, these studies establish the blockade of glycolysis at the Glyceraldehyde 3-phosphate dehydrogenase step as the central metabolic basis of NAMPT inhibition responsible for ATP depletion, metabolic perturbation, and subsequent tumor growth inhibition. These studies also suggest that altered metabolite levels in tumors can be used as robust pharmacodynamic markers for evaluating NAMPT inhibitors in the clinic. Background: NAMPT catalyzes the rate-limiting reaction in converting nicotinamide to NAD+ in cancers. Results: NAMPT inhibition attenuates glycolysis at the Glyceraldehyde 3-phosphate dehydrogenase step, resulting in perturbing metabolic pathways related to glycolysis. Conclusion: The metabolic basis of NAMPT inhibition is the attenuation of glycolysis by reducing NAD+ available to Glyceraldehyde 3-phosphate dehydrogenase. Significance: This study sheds new light on how NAMPT regulates cancer metabolism.
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pharmacological inhibition of nicotinamide phosphoribosyltransferase nampt an enzyme essential for nad biosynthesis in human cancer cells metabolic basis and potential clinical implications
Journal of Biological Chemistry, 2013Co-Authors: Debra A Young, Robert L Shepard, K Roth, Tao Wang, Timothy I Meier, Yan Zhai, Karen L Huss, James Ronald Gillig, Zhao Hai Lu, Saravanan ParthasarathyAbstract:Abstract Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the first rate-limiting step in converting nicotinamide to NAD+, essential for cellular metabolism, energy production, and DNA repair. NAMPT has been extensively studied because of its critical role in these cellular processes and the prospect of developing therapeutics against the target, yet how it regulates cellular metabolism is not fully understood. In this study we utilized liquid chromatography-mass spectrometry to examine the effects of FK866, a small molecule inhibitor of NAMPT currently in clinical trials, on glycolysis, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, and serine biosynthesis in cancer cells and tumor xenografts. We show for the first time that NAMPT inhibition leads to the attenuation of glycolysis at the Glyceraldehyde 3-phosphate dehydrogenase step due to the reduced availability of NAD+ for the enzyme. The attenuation of glycolysis results in the accumulation of glycolytic intermediates before and at the Glyceraldehyde 3-phosphate dehydrogenase step, promoting carbon overflow into the pentose phosphate pathway as evidenced by the increased intermediate levels. The attenuation of glycolysis also causes decreased glycolytic intermediates after the Glyceraldehyde 3-phosphate dehydrogenase step, thereby reducing carbon flow into serine biosynthesis and the TCA cycle. Labeling studies establish that the carbon overflow into the pentose phosphate pathway is mainly through its non-oxidative branch. Together, these studies establish the blockade of glycolysis at the Glyceraldehyde 3-phosphate dehydrogenase step as the central metabolic basis of NAMPT inhibition responsible for ATP depletion, metabolic perturbation, and subsequent tumor growth inhibition. These studies also suggest that altered metabolite levels in tumors can be used as robust pharmacodynamic markers for evaluating NAMPT inhibitors in the clinic.
Robert L Shepard - One of the best experts on this subject based on the ideXlab platform.
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inhibition of nicotinamide phosphoribosyltransferase nampt an enzyme essential for nad biosynthesis leads to altered carbohydrate metabolism in cancer cells
Journal of Biological Chemistry, 2015Co-Authors: Sucai Dong, Robert L Shepard, Lisa Kays, K Roth, Sandaruwan Geeganage, Genshi ZhaoAbstract:Abstract NAMPT has been extensively studied due to its essential role in NAD+ biosynthesis in cancer cells and prospect for developing novel therapeutics. To understand how NAMPT regulates cellular metabolism, we have shown that the treatment with FK866, a specific NAMPT inhibitor, leads to attenuation of glycolysis by blocking the Glyceraldehyde 3-phosphate dehydrogenase step (1). Due to technical limitations, we failed to separate isotopmers of phosphorylated sugars. In this study, we developed an enabling LC-MS methodology. Using this, we confirmed the previous findings (1) and also showed that NAMPT inhibition led to accumulation of fructose 1-phosphate and sedoheptulose 1-phosphate, but not glucose 6-phosphate, fructose 6-phosphate, and sedoheptulose 7-phosphate as previously thought. To investigate the metabolic basis of the metabolite formation, we carried out biochemical and cellular studies and established the following. First, glucose-labeling studies indicated that fructose 1-phosphate was derived from dihydroxyacetone phosphate and Glyceraldehyde, and sedoheptulose 1-phosphate from dihydroxyacetone phosphate and erythrose via an aldolase reaction. Second, biochemical studies showed that aldolase indeed catalyzed these reactions. Third, Glyceraldehyde- and erythrose-labeling studies showed increased incorporation of corresponding labels into fructose 1-phosphate and sedoheptulose 1-phosphate in FK866-treated cells. Fourth, NAMPT inhibition led to increased Glyceraldehyde and erythrose levels in the cell. Finally, glucose-labeling studies showed accumulated fructose 1,6-bisphosphate in FK866-treated cells mainly derived from dihydroxyacetone phosphate and Glyceraldehyde 3-phosphate. Taken together, this study shows that NAMPT inhibition leads to attenuation of glycolysis, resulting in further perturbation of carbohydrate metabolism in cancer cells. The potential clinical implications of these findings are also discussed.
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pharmacological inhibition of nicotinamide phosphoribosyltransferase nampt an enzyme essential for nad biosynthesis in human cancer cells metabolic basis and potential clinical implications
Journal of Biological Chemistry, 2013Co-Authors: Bo Tan, Robert L Shepard, K Roth, Debra A Young, Tao Wang, Timothy I Meier, Yan Zhai, Karen L Huss, Mingshang Kuo, James Ronald GilligAbstract:Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the first rate-limiting step in converting nicotinamide to NAD+, essential for cellular metabolism, energy production, and DNA repair. NAMPT has been extensively studied because of its critical role in these cellular processes and the prospect of developing therapeutics against the target, yet how it regulates cellular metabolism is not fully understood. In this study we utilized liquid chromatography-mass spectrometry to examine the effects of FK866, a small molecule inhibitor of NAMPT currently in clinical trials, on glycolysis, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, and serine biosynthesis in cancer cells and tumor xenografts. We show for the first time that NAMPT inhibition leads to the attenuation of glycolysis at the Glyceraldehyde 3-phosphate dehydrogenase step due to the reduced availability of NAD+ for the enzyme. The attenuation of glycolysis results in the accumulation of glycolytic intermediates before and at the Glyceraldehyde 3-phosphate dehydrogenase step, promoting carbon overflow into the pentose phosphate pathway as evidenced by the increased intermediate levels. The attenuation of glycolysis also causes decreased glycolytic intermediates after the Glyceraldehyde 3-phosphate dehydrogenase step, thereby reducing carbon flow into serine biosynthesis and the TCA cycle. Labeling studies establish that the carbon overflow into the pentose phosphate pathway is mainly through its non-oxidative branch. Together, these studies establish the blockade of glycolysis at the Glyceraldehyde 3-phosphate dehydrogenase step as the central metabolic basis of NAMPT inhibition responsible for ATP depletion, metabolic perturbation, and subsequent tumor growth inhibition. These studies also suggest that altered metabolite levels in tumors can be used as robust pharmacodynamic markers for evaluating NAMPT inhibitors in the clinic. Background: NAMPT catalyzes the rate-limiting reaction in converting nicotinamide to NAD+ in cancers. Results: NAMPT inhibition attenuates glycolysis at the Glyceraldehyde 3-phosphate dehydrogenase step, resulting in perturbing metabolic pathways related to glycolysis. Conclusion: The metabolic basis of NAMPT inhibition is the attenuation of glycolysis by reducing NAD+ available to Glyceraldehyde 3-phosphate dehydrogenase. Significance: This study sheds new light on how NAMPT regulates cancer metabolism.
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pharmacological inhibition of nicotinamide phosphoribosyltransferase nampt an enzyme essential for nad biosynthesis in human cancer cells metabolic basis and potential clinical implications
Journal of Biological Chemistry, 2013Co-Authors: Debra A Young, Robert L Shepard, K Roth, Tao Wang, Timothy I Meier, Yan Zhai, Karen L Huss, James Ronald Gillig, Zhao Hai Lu, Saravanan ParthasarathyAbstract:Abstract Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the first rate-limiting step in converting nicotinamide to NAD+, essential for cellular metabolism, energy production, and DNA repair. NAMPT has been extensively studied because of its critical role in these cellular processes and the prospect of developing therapeutics against the target, yet how it regulates cellular metabolism is not fully understood. In this study we utilized liquid chromatography-mass spectrometry to examine the effects of FK866, a small molecule inhibitor of NAMPT currently in clinical trials, on glycolysis, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, and serine biosynthesis in cancer cells and tumor xenografts. We show for the first time that NAMPT inhibition leads to the attenuation of glycolysis at the Glyceraldehyde 3-phosphate dehydrogenase step due to the reduced availability of NAD+ for the enzyme. The attenuation of glycolysis results in the accumulation of glycolytic intermediates before and at the Glyceraldehyde 3-phosphate dehydrogenase step, promoting carbon overflow into the pentose phosphate pathway as evidenced by the increased intermediate levels. The attenuation of glycolysis also causes decreased glycolytic intermediates after the Glyceraldehyde 3-phosphate dehydrogenase step, thereby reducing carbon flow into serine biosynthesis and the TCA cycle. Labeling studies establish that the carbon overflow into the pentose phosphate pathway is mainly through its non-oxidative branch. Together, these studies establish the blockade of glycolysis at the Glyceraldehyde 3-phosphate dehydrogenase step as the central metabolic basis of NAMPT inhibition responsible for ATP depletion, metabolic perturbation, and subsequent tumor growth inhibition. These studies also suggest that altered metabolite levels in tumors can be used as robust pharmacodynamic markers for evaluating NAMPT inhibitors in the clinic.
K Roth - One of the best experts on this subject based on the ideXlab platform.
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inhibition of nicotinamide phosphoribosyltransferase nampt an enzyme essential for nad biosynthesis leads to altered carbohydrate metabolism in cancer cells
Journal of Biological Chemistry, 2015Co-Authors: Sucai Dong, Robert L Shepard, Lisa Kays, K Roth, Sandaruwan Geeganage, Genshi ZhaoAbstract:Abstract NAMPT has been extensively studied due to its essential role in NAD+ biosynthesis in cancer cells and prospect for developing novel therapeutics. To understand how NAMPT regulates cellular metabolism, we have shown that the treatment with FK866, a specific NAMPT inhibitor, leads to attenuation of glycolysis by blocking the Glyceraldehyde 3-phosphate dehydrogenase step (1). Due to technical limitations, we failed to separate isotopmers of phosphorylated sugars. In this study, we developed an enabling LC-MS methodology. Using this, we confirmed the previous findings (1) and also showed that NAMPT inhibition led to accumulation of fructose 1-phosphate and sedoheptulose 1-phosphate, but not glucose 6-phosphate, fructose 6-phosphate, and sedoheptulose 7-phosphate as previously thought. To investigate the metabolic basis of the metabolite formation, we carried out biochemical and cellular studies and established the following. First, glucose-labeling studies indicated that fructose 1-phosphate was derived from dihydroxyacetone phosphate and Glyceraldehyde, and sedoheptulose 1-phosphate from dihydroxyacetone phosphate and erythrose via an aldolase reaction. Second, biochemical studies showed that aldolase indeed catalyzed these reactions. Third, Glyceraldehyde- and erythrose-labeling studies showed increased incorporation of corresponding labels into fructose 1-phosphate and sedoheptulose 1-phosphate in FK866-treated cells. Fourth, NAMPT inhibition led to increased Glyceraldehyde and erythrose levels in the cell. Finally, glucose-labeling studies showed accumulated fructose 1,6-bisphosphate in FK866-treated cells mainly derived from dihydroxyacetone phosphate and Glyceraldehyde 3-phosphate. Taken together, this study shows that NAMPT inhibition leads to attenuation of glycolysis, resulting in further perturbation of carbohydrate metabolism in cancer cells. The potential clinical implications of these findings are also discussed.
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pharmacological inhibition of nicotinamide phosphoribosyltransferase nampt an enzyme essential for nad biosynthesis in human cancer cells metabolic basis and potential clinical implications
Journal of Biological Chemistry, 2013Co-Authors: Bo Tan, Robert L Shepard, K Roth, Debra A Young, Tao Wang, Timothy I Meier, Yan Zhai, Karen L Huss, Mingshang Kuo, James Ronald GilligAbstract:Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the first rate-limiting step in converting nicotinamide to NAD+, essential for cellular metabolism, energy production, and DNA repair. NAMPT has been extensively studied because of its critical role in these cellular processes and the prospect of developing therapeutics against the target, yet how it regulates cellular metabolism is not fully understood. In this study we utilized liquid chromatography-mass spectrometry to examine the effects of FK866, a small molecule inhibitor of NAMPT currently in clinical trials, on glycolysis, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, and serine biosynthesis in cancer cells and tumor xenografts. We show for the first time that NAMPT inhibition leads to the attenuation of glycolysis at the Glyceraldehyde 3-phosphate dehydrogenase step due to the reduced availability of NAD+ for the enzyme. The attenuation of glycolysis results in the accumulation of glycolytic intermediates before and at the Glyceraldehyde 3-phosphate dehydrogenase step, promoting carbon overflow into the pentose phosphate pathway as evidenced by the increased intermediate levels. The attenuation of glycolysis also causes decreased glycolytic intermediates after the Glyceraldehyde 3-phosphate dehydrogenase step, thereby reducing carbon flow into serine biosynthesis and the TCA cycle. Labeling studies establish that the carbon overflow into the pentose phosphate pathway is mainly through its non-oxidative branch. Together, these studies establish the blockade of glycolysis at the Glyceraldehyde 3-phosphate dehydrogenase step as the central metabolic basis of NAMPT inhibition responsible for ATP depletion, metabolic perturbation, and subsequent tumor growth inhibition. These studies also suggest that altered metabolite levels in tumors can be used as robust pharmacodynamic markers for evaluating NAMPT inhibitors in the clinic. Background: NAMPT catalyzes the rate-limiting reaction in converting nicotinamide to NAD+ in cancers. Results: NAMPT inhibition attenuates glycolysis at the Glyceraldehyde 3-phosphate dehydrogenase step, resulting in perturbing metabolic pathways related to glycolysis. Conclusion: The metabolic basis of NAMPT inhibition is the attenuation of glycolysis by reducing NAD+ available to Glyceraldehyde 3-phosphate dehydrogenase. Significance: This study sheds new light on how NAMPT regulates cancer metabolism.
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pharmacological inhibition of nicotinamide phosphoribosyltransferase nampt an enzyme essential for nad biosynthesis in human cancer cells metabolic basis and potential clinical implications
Journal of Biological Chemistry, 2013Co-Authors: Debra A Young, Robert L Shepard, K Roth, Tao Wang, Timothy I Meier, Yan Zhai, Karen L Huss, James Ronald Gillig, Zhao Hai Lu, Saravanan ParthasarathyAbstract:Abstract Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the first rate-limiting step in converting nicotinamide to NAD+, essential for cellular metabolism, energy production, and DNA repair. NAMPT has been extensively studied because of its critical role in these cellular processes and the prospect of developing therapeutics against the target, yet how it regulates cellular metabolism is not fully understood. In this study we utilized liquid chromatography-mass spectrometry to examine the effects of FK866, a small molecule inhibitor of NAMPT currently in clinical trials, on glycolysis, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, and serine biosynthesis in cancer cells and tumor xenografts. We show for the first time that NAMPT inhibition leads to the attenuation of glycolysis at the Glyceraldehyde 3-phosphate dehydrogenase step due to the reduced availability of NAD+ for the enzyme. The attenuation of glycolysis results in the accumulation of glycolytic intermediates before and at the Glyceraldehyde 3-phosphate dehydrogenase step, promoting carbon overflow into the pentose phosphate pathway as evidenced by the increased intermediate levels. The attenuation of glycolysis also causes decreased glycolytic intermediates after the Glyceraldehyde 3-phosphate dehydrogenase step, thereby reducing carbon flow into serine biosynthesis and the TCA cycle. Labeling studies establish that the carbon overflow into the pentose phosphate pathway is mainly through its non-oxidative branch. Together, these studies establish the blockade of glycolysis at the Glyceraldehyde 3-phosphate dehydrogenase step as the central metabolic basis of NAMPT inhibition responsible for ATP depletion, metabolic perturbation, and subsequent tumor growth inhibition. These studies also suggest that altered metabolite levels in tumors can be used as robust pharmacodynamic markers for evaluating NAMPT inhibitors in the clinic.
Patrik R Jones - One of the best experts on this subject based on the ideXlab platform.
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Glyceraldehyde 3 phosphate ferredoxin oxidoreductase from methanococcus maripaludis
Journal of Bacteriology, 2007Co-Authors: Myongok Park, Taeko Mizutani, Patrik R JonesAbstract:The genome sequence of the non-sugar-assimilating mesophile Methanococcus maripaludis contains three genes encoding enzymes: a nonphosphorylating NADP+-dependent Glyceraldehyde-3-phosphate dehydrogenase (GAPN), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and Glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR); all these enzymes are potentially capable of catalyzing Glyceraldehyde-3-phosphate (G3P) metabolism. GAPOR, whose homologs have been found mainly in archaea, catalyzes the reduction of ferredoxin coupled with oxidation of G3P. GAPOR has previously been isolated and characterized only from a sugar-assimilating hyperthermophile, Pyrococcus furiosus (GAPORPf), and contains the rare metal tungsten as an irreplaceable cofactor. Active recombinant M. maripaludis GAPOR (GAPORMm) was purified from Escherichia coli grown in minimal medium containing 100 μM sodium molybdate. In contrast, GAPORMm obtained from cells grown in medium containing tungsten (W) and W and molybdenum (Mo) or in medium without added W and Mo did not display any activity. Activity and transcript analysis of putative G3P-metabolizing enzymes and corresponding genes were performed with M. maripaludis cultured under autotrophic conditions in chemically defined medium. The activity of GAPORMm was constitutive throughout the culture period and exceeded that of GAPDH at all time points. As GAPDH activity was detected in only the gluconeogenic direction and GAPN activity was completely absent, only GAPORMm catalyzes oxidation of G3P in M. maripaludis. Recombinant GAPORMm is posttranscriptionally regulated as it exhibits pronounced and irreversible substrate inhibition and is completely inhibited by 1 μM ATP. With support from flux balance analysis, it is concluded that the major physiological role of GAPORMm in M. maripaludis most likely involves only nonoptimal growth conditions.
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Glyceraldehyde 3 phosphate ferredoxin oxidoreductase from methanococcus maripaludis
Journal of Bacteriology, 2007Co-Authors: Myongok Park, Taeko Mizutani, Patrik R JonesAbstract:The genome sequence of the non-sugar-assimilating mesophile Methanococcus maripaludis contains three genes encoding enzymes: a nonphosphorylating NADP+-dependent Glyceraldehyde-3-phosphate dehydrogenase (GAPN), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and Glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR); all these enzymes are potentially capable of catalyzing Glyceraldehyde-3-phosphate (G3P) metabolism. GAPOR, whose homologs have been found mainly in archaea, catalyzes the reduction of ferredoxin coupled with oxidation of G3P. GAPOR has previously been isolated and characterized only from a sugar-assimilating hyperthermophile, Pyrococcus furiosus (GAPORPf), and contains the rare metal tungsten as an irreplaceable cofactor. Active recombinant M. maripaludis GAPOR (GAPORMm) was purified from Escherichia coli grown in minimal medium containing 100 μM sodium molybdate. In contrast, GAPORMm obtained from cells grown in medium containing tungsten (W) and W and molybdenum (Mo) or in medium without added W and Mo did not display any activity. Activity and transcript analysis of putative G3P-metabolizing enzymes and corresponding genes were performed with M. maripaludis cultured under autotrophic conditions in chemically defined medium. The activity of GAPORMm was constitutive throughout the culture period and exceeded that of GAPDH at all time points. As GAPDH activity was detected in only the gluconeogenic direction and GAPN activity was completely absent, only GAPORMm catalyzes oxidation of G3P in M. maripaludis. Recombinant GAPORMm is posttranscriptionally regulated as it exhibits pronounced and irreversible substrate inhibition and is completely inhibited by 1 μM ATP. With support from flux balance analysis, it is concluded that the major physiological role of GAPORMm in M. maripaludis most likely involves only nonoptimal growth conditions.