The Experts below are selected from a list of 243 Experts worldwide ranked by ideXlab platform
Lizbeth Hedstrom - One of the best experts on this subject based on the ideXlab platform.
-
Allosteric activation via kinetic control: Potassium accelerates a conformational change in IMP Dehydrogenase
Biochemistry, 2011Co-Authors: Thomas V. Riera, Lianqing Zheng, Helen R. Josephine, Donghong Min, Wei Yang, Lizbeth HedstromAbstract:Allosteric activators are generally believed to shift the equilibrium distribution of enzyme conformations to favor a catalytically productive structure; the kinetics of conformational exchange is seldom addressed. Several observations suggested that the usual allosteric mechanism might not apply to the activation of IMP Dehydrogenase (IMPDH) by monovalent cations. Therefore, we investigated the mechanism of K(+) activation in IMPDH by delineating the kinetic mechanism in the absence of monovalent cations. Surprisingly, the K(+) dependence of k(cat) derives from the rate of flap closure, which increases by ≥65-fold in the presence of K(+). We performed both alchemical free energy simulations and potential of mean force calculations using the orthogonal space random walk strategy to computationally analyze how K(+) accelerates this conformational change. The simulations recapitulate the preference of IMPDH for K(+), validating the computational models. When K(+) is replaced with a dummy ion, the residues of the K(+) binding site relax into ordered secondary structure, creating a barrier to conformational exchange. K(+) mobilizes these residues by providing alternate interactions for the main chain carbonyls. Potential of mean force calculations indicate that K(+) changes the shape of the energy well, shrinking the reaction coordinate by shifting the closed conformation toward the open state. This work suggests that allosteric regulation can be under kinetic as well as thermodynamic control.
-
Specific biotinylation of IMP Dehydrogenase.
Bioorganic & medicinal chemistry letters, 2011Co-Authors: B. Christopher Hoefler, Deviprasad R. Gollapalli, Lizbeth HedstromAbstract:IMP Dehydrogenase (IMPDH) catalyzes a critical step in guanine nucleotide biosynthesis. IMPDH also has biological roles that are distinct from its enzymatic function. We report a biotin-linked reagent that selectively labels IMPDH and is released by dithiothreitol. This reagent will be invaluable in elucidating the moonlighting functions of IMPDH.
-
IMP Dehydrogenase type 1 associates with polyribosomes translating rhodopsin mRNA.
The Journal of biological chemistry, 2008Co-Authors: Sarah E. Mortimer, Sara J Bowne, Stephen P Daiger, Dharia A. Mcgrew, Nobuko Hamaguchi, Hoong Chuin Lim, Lizbeth HedstromAbstract:IMP Dehydrogenase (IMPDH) catalyzes the pivotal step in guanine nucleotide biosynthesis. Here we show that both IMPDH type 1 (IMPDH1) and IMPDH type 2 are associated with polyribosomes, suggesting that these housekeeping proteins have an unanticipated role in translation regulation. This interaction is mediated by the subdomain, a region of disputed function that is the site of mutations that cause retinal degeneration. The retinal isoforms of IMPDH1 also associate with polyribosomes. The most common disease-causing mutation, D226N, disrupts the polyribosome association of at least one retinal IMPDH1 isoform. Finally, we find that IMPDH1 is associated with polyribosomes containing rhodopsin mRNA. Because any perturbation of rhodopsin expression can trigger apoptosis in photoreceptor cells, these observations suggest a likely pathological mechanism for IMPDH1-mediated hereditary blindness. We propose that IMPDH coordinates the translation of a set of mRNAs, perhaps by modulating localization or degradation.
-
Guanidine derivatives rescue the Arg418Ala mutation of Tritrichomonas foetus IMP Dehydrogenase.
Biochemistry, 2005Co-Authors: Yollete V. Guillen Schlippe, Lizbeth HedstromAbstract:IMP Dehydrogenase (IMPDH) catalyzes the oxidation of inosine 5‘-monophosphate (IMP) to xanthosine 5‘-monophosphate (XMP) and the reduction of NAD+. The reaction involves formation of an E−XMP* covalent intermediate; hydrolysis of the E−XMP* intermediate is rate-limiting and requires the enzyme to adopt a closed conformation. Arg418 appears to act as the base that activates water for the hydrolysis reaction [Guillen-Schlippe, Y. V., and Hedstrom, L. (2005) Biochemistry 44, 11700−11707]. Deprotonation of Arg418 also stabilizes the closed conformation. Here we show that guanidine derivatives rescue the activity of the Arg418Ala variant. Amines and imidazole do not rescue. The rescue reaction appears to be saturable, with the values of KR ranging from 40 to 400 mM. The value of krescue for the best rescue agents approaches the value of kcat for the reaction of the wild-type enzyme. Guanidine derivatives also rescue the activity of the Arg418Ala/Tyr419Phe variant. Multiple-inhibitor experiments suggest that th...
-
Is Arg418 the catalytic base required for the hydrolysis step of the IMP Dehydrogenase reaction
Biochemistry, 2005Co-Authors: Yollete V. Guillen Schlippe, Lizbeth HedstromAbstract:The first committed step of guanine nucleotide biosynthesis is the oxidation of inosine 5‘-monophosphate (IMP) to xanthosine 5‘-monophosphate (XMP) catalyzed by IMP Dehydrogenase. The reaction involves the reduction of NAD+ with the formation of a covalent enzyme intermediate (E−XMP*). Hydrolysis of E−XMP* requires the enzyme to adopt a closed conformation and is rate-limiting. Thr321, Arg418, and Tyr419 are candidates for the residue that activates water. The substitution of Thr321 has similar, but small, effects on both the hydride transfer and hydrolysis steps. This result suggests that Thr321 influences the reactivity of Cys319, either through a direct interaction or by stabilizing the structure of the active site loop. The hydrolysis of E−XMP* is accelerated by the deprotonation of a residue with a pKa of ∼8. A similar deprotonation stabilizes the closed conformation; this residue has a pKa of ≥6 in the closed conformation. The substitution of Tyr419 with Phe does not change the pH dependence of eith...
George D Markham - One of the best experts on this subject based on the ideXlab platform.
-
A Regulatory Role of the Bateman Domain of IMP Dehydrogenase in Adenylate Nucleotide Biosynthesis
The Journal of biological chemistry, 2009Co-Authors: Maxim Pimkin, Julia Pimkina, George D MarkhamAbstract:The Bateman domain (CBS subdomain) of IMP Dehydrogenase (IMPDH), a rate-limiting enzyme of the de novo GMP biosynthesis, is evolutionarily conserved but has no established function. Deletion of the Bateman domain has no effect on the in vitro IMPDH activity. We report that in vivo deletion of the Bateman domain of IMPDH in Escherichia coli (guaBΔCBS) sensitizes the bacterium to growth arrest by adenosine and inosine. These nucleosides exert their growth inhibitory effect via a dramatic increase in the intracellular adenylate nucleotide pool, which results in the enhanced allosteric inhibition of PRPP synthetase and consequently a PRPP deficit. The ensuing starvation for pyrimidine nucleotides culminates in growth arrest. Thus, deletion of the Bateman domain of IMPDH derepresses the synthesis of AMP from IMP. The growth inhibitory effect of inosine can be rescued by second-site suppressor mutations in the genes responsible for the conversion of inosine to AMP (gsk, purA, and purB) as well as by the prsA1 allele, which encodes a PRPP synthetase that is insensitive to allosteric inhibition by adenylate nucleotides. IMPortantly, the guaBΔCBS phenotype can be complemented in trans by a mutant guaB allele, which encodes a catalytically disabled IMPDHC305A protein containing an intact Bateman domain. We conclude that the Bateman domain of IMPDH is a negative trans-regulator of adenylate nucleotide synthesis, and that this role is independent of the catalytic function of IMPDH in the de novo GMP biosynthesis.
-
A REGULATORY ROLE OF THE BATEMAN DOMAIN OF IMP Dehydrogenase IN
2009Co-Authors: Adenylate Nucleotide Biosynthesis, Maxim Pimkin, Julia Pimkina, George D MarkhamAbstract:The Bateman domain (CBS subdomain) of IMP Dehydrogenase (IMPDH), a rate-limiting enzyme of the de novo GMP biosynthesis, is evolutionarily conserved but has no established function. Deletion of the Bateman domain has no effect on the in vitro IMPDH activity. We report that in vivo deletion of the Bateman domain of IMPDH in Escherichia coli (guaB ∆CBS ) sensitizes the bacterium to growth arrest by adenosine and inosine. These nucleosides exert their growth inhibitory effect via a dramatic increase in the intracellular adenylate nucleotide pool, which results in the enhanced allosteric inhibition of PRPP synthetase and consequently a PRPP deficit. The ensuing starvation for pyrimidine nucleotides culminates in growth arrest. Thus, deletion of the Bateman domain of IMPDH derepresses the synthesis of AMP from IMP. The growth inhibitory effect of inosine can be rescued by second-site suppressor mutations in the genes responsible for the conversion of inosine to AMP (gsk, purA, purB) as well as by the prsA1 allele which encodes a PRPP synthetase that is insensitive to allosteric inhibition by adenylate nucleotides. IMPortantly, the guaB ∆CBS phenotype can be complemented in trans by a mutant guaB allele which encodes a catalytically disabled IMPDH C305A protein containing an intact Bateman domain. We conclude that the Bateman domain of IMPDH is a negative trans- regulator of adenylate nucleotide synthesis, and that this role is independent of the catalytic function of IMPDH in the de novo GMP biosynthesis.
-
the conformation of inosine 5 monophosphate IMP bound to IMP Dehydrogenase determined by transferred nuclear overhauser effect spectroscopy
Journal of Biological Chemistry, 1996Co-Authors: Bosong Xiang, George D MarkhamAbstract:Abstract IMP Dehydrogenase (IMPDH) catalyzes the NAD-dependent synthesis of xanthosine 5′-monophosphate which is the rate-limiting step in guanine nucleotide biosynthesis. Although IMPDH is the target of numerous chemotherapeutic agents, nothing has been known about the conformation of the enzyme-bound substrates. The conformation of IMP bound to human type II IMP Dehydrogenase has been determined by two-dimensional transferred nuclear Overhauser effect NMR spectroscopy at 600 MHz. NOE buildup rates were determined by recording NOESY spectra at numerous mixing times. The cross-relaxation rates determined from the initial NOE build-up rates were used to calculate inter-proton distances of bound IMP. The conformation of the enzyme-bound IMP was obtained by molecular modeling with energy minimization using the experimentally determined inter-proton distance constraints. The glycosidic torsion angle of the bound nucleotide is anti and the sugar is in the C2-endo-conformation. This conformation places H2 of IMP, which is transferred to NAD in the reaction, in a position clear of the rest of the molecule in order to facilitate the reaction.
-
The Conformation of Inosine 5′-Monophosphate (IMP) Bound to IMP Dehydrogenase Determined by Transferred Nuclear Overhauser Effect Spectroscopy
The Journal of biological chemistry, 1996Co-Authors: Bosong Xiang, George D MarkhamAbstract:Abstract IMP Dehydrogenase (IMPDH) catalyzes the NAD-dependent synthesis of xanthosine 5′-monophosphate which is the rate-limiting step in guanine nucleotide biosynthesis. Although IMPDH is the target of numerous chemotherapeutic agents, nothing has been known about the conformation of the enzyme-bound substrates. The conformation of IMP bound to human type II IMP Dehydrogenase has been determined by two-dimensional transferred nuclear Overhauser effect NMR spectroscopy at 600 MHz. NOE buildup rates were determined by recording NOESY spectra at numerous mixing times. The cross-relaxation rates determined from the initial NOE build-up rates were used to calculate inter-proton distances of bound IMP. The conformation of the enzyme-bound IMP was obtained by molecular modeling with energy minimization using the experimentally determined inter-proton distance constraints. The glycosidic torsion angle of the bound nucleotide is anti and the sugar is in the C2-endo-conformation. This conformation places H2 of IMP, which is transferred to NAD in the reaction, in a position clear of the rest of the molecule in order to facilitate the reaction.
Daniel Reines - One of the best experts on this subject based on the ideXlab platform.
-
Detection of the mycophenolate-inhibited form of IMP Dehydrogenase in vivo
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Christine C. Mcphillips, Judith W. Hyle, Daniel ReinesAbstract:IMP Dehydrogenase (IMPDH) is the rate-limiting enzyme for de novo GMP synthesis. Its activity is correlated with cell growth, and it is the target of a number of proven and experimental drug therapies including mycophenolic acid (MPA). MPA inhibits the enzyme by trapping a covalent nucleotide-enzyme intermediate. Saccharomyces cerevisiae has four IMPDH genes called IMD1—IMD4. IMD2 is transcriptionally regulated and is the only one that enables yeast to grow in the presence of MPA. We show here that de novo synthesis of the IMD2-encoded protein is strongly induced upon MPA treatment. We also monitor the in vivo formation of a covalent nucleotide-enzyme intermediate for Imd2, Imd3, and Imd4 that accumulates in the presence of MPA. Complete formation of the Imd2 intermediate requires drug concentrations manyfold higher than that required to quantitatively trap the Imd3- or Imd4-nucleotide adducts. Purification of the tagged IMD gene products reveals that the family of polypeptides coassemble to form heteromeric IMPDH complexes, suggesting that they form mixed tetramers. These data demonstrate that S. cerevisiae harbor multiple IMPDH enzymes with varying drug sensitivities and offer an assay to monitor the inhibition of IMPDH in living cells. They also suggest that mixed inhibition profiles may result from heteromeric complexes in cell types that contain multiple IMPDH gene products. The mobility shift assay could serve as a tool for the detection of drug-inactivated IMPDH in the cells of patients receiving MPA therapy.
-
Large‐scale screening of yeast mutants for sensitivity to the IMP Dehydrogenase inhibitor 6‐azauracil
Yeast (Chichester England), 2004Co-Authors: Linda Riles, Randal J Shaw, Mark Johnston, Daniel ReinesAbstract:Mutations in several genes encoding components of the RNA polymerase II elongation machinery render S. cerevisiae cells sensitive to the drug 6-azauracil (6AU), an inhibitor of IMP Dehydrogenase and orotidylate decarboxylase. It is thought that a reduction in nucleotide levels following drug treatment causes transcriptional elongation to be more dependent on a fully functional RNA polymerase. To gain insight into the basis of the 6AU-sensitive phenotype and discern its specificity, we screened almost 3000 deletion mutants for growth in the presence of drug; 42 (1.5%) were reproducibly sensitive to the drug. The sensitive mutants included several missing known transcription elongation factors, but the majority were in genes involved in other cellular processes. Not all of the 6AU-sensitive strains displayed cross-sensitivity to mycophenolic acid (MPA), another drug that inhibits IMP Dehydrogenase and has been employed as a screening agent for elongation mutants, showing that these two drugs are mechanistically distinct. Several of the mutants were tested for the ability to induce transcription of IMP Dehydrogenase-encoding genes, in response to 6-AU and MPA treatment. As expected, mutants defective in transcriptional elongation factors were unable to fully induce IMPDH expression. However, most of the 6AU-sensitive strains had normal levels of IMPDH expression. Thus, although 6AU-sensitivity often results from defects in the elongation machinery, mutations that compromise processes other than transcription and induction of IMPDH also lead to sensitivity to this drug.
-
large scale screening of yeast mutants for sensitivity to the IMP Dehydrogenase inhibitor 6 azauracil
Yeast, 2004Co-Authors: Linda Riles, Randal J Shaw, Mark Johnston, Daniel ReinesAbstract:Mutations in several genes encoding components of the RNA polymerase II elongation machinery render S. cerevisiae cells sensitive to the drug 6-azauracil (6AU), an inhibitor of IMP Dehydrogenase and orotidylate decarboxylase. It is thought that a reduction in nucleotide levels following drug treatment causes transcriptional elongation to be more dependent on a fully functional RNA polymerase. To gain insight into the basis of the 6AU-sensitive phenotype and discern its specificity, we screened almost 3000 deletion mutants for growth in the presence of drug; 42 (1.5%) were reproducibly sensitive to the drug. The sensitive mutants included several missing known transcription elongation factors, but the majority were in genes involved in other cellular processes. Not all of the 6AU-sensitive strains displayed cross-sensitivity to mycophenolic acid (MPA), another drug that inhibits IMP Dehydrogenase and has been employed as a screening agent for elongation mutants, showing that these two drugs are mechanistically distinct. Several of the mutants were tested for the ability to induce transcription of IMP Dehydrogenase-encoding genes, in response to 6-AU and MPA treatment. As expected, mutants defective in transcriptional elongation factors were unable to fully induce IMPDH expression. However, most of the 6AU-sensitive strains had normal levels of IMPDH expression. Thus, although 6AU-sensitivity often results from defects in the elongation machinery, mutations that compromise processes other than transcription and induction of IMPDH also lead to sensitivity to this drug.
-
Functional distinctions between IMP Dehydrogenase genes in providing mycophenolate resistance and guanine prototrophy to yeast
The Journal of biological chemistry, 2003Co-Authors: Judith W. Hyle, Randal J Shaw, Daniel ReinesAbstract:IMP Dehydrogenase (IMPDH) catalyzes the rate-limiting step in the de novo synthesis of GTP. Yeast with mutations in the transcription elongation machinery are sensitive to inhibitors of this enzyme such as 6-azauracil and mycophenolic acid, at least partly because of their inability to transcriptionally induce IMPDH. To understand the molecular basis of this drug-sensitive phenotype, we have dissected the expression and function of a four-gene family in yeast called IMD1 through IMD4. We show here that these family members are distinct, despite a high degree of amino acid identity between the proteins they encode. Extrachromosomal copies of IMD1, IMD3, or IMD4 could not rescue the drug-sensitive phenotype of IMD2 deletants. When overexpressed, IMD3 or IMD4 weakly compensated for deletion of IMD2. IMD1 is transcriptionally silent and bears critical amino acid substitutions compared with IMD2 that destroy its function, offering strong evidence that it is a pseudogene. The simultaneous deletion of all four IMD genes was lethal unless growth media were supplemented with guanine. This suggests that there are no other essential functions of the IMPDH homologs aside from IMP Dehydrogenase activity. Although neither IMD3 nor IMD4 could confer drug resistance to cells lacking IMD2, either alone was sufficient to confer guanine prototrophy. The special function of IMD2 was provided by its ability to be transcriptionally induced and the probable intrinsic drug resistance of its enzymatic activity.
-
Regulation of an IMP Dehydrogenase Gene and Its Overexpression in Drug-sensitive Transcription Elongation Mutants of Yeast
The Journal of biological chemistry, 2001Co-Authors: Randal J Shaw, Judith L. Wilson, Karen T. Smith, Daniel ReinesAbstract:Abstract IMP Dehydrogenase is a rate-limiting enzyme involved in the synthesis of GTP. In mammalian cells it is regulated with respect to growth rate and is the target of numerous therapeutic agents. Mutations in the RNA polymerase II elongation machinery render yeast sensitive to inhibitors of IMP Dehydrogenase and defective in inducing transcription of one of the IMP Dehydrogenase-encoding genes,IMD2. Here we show that loss of IMD2, but notIMD1, IMD3, or IMD4, conferred upon yeast the same drug sensitivity found in elongation mutants. We tested whether the drug sensitivity of elongation mutants is due to their inability to induce IMD2 by providing them with exogenous copies of the gene. In some elongation mutants, overexpression reversed drug sensitivity and a transcriptional defect. Overexpression in mutants with a more severe phenotype partially suppressed drug sensitivity but was inconsequential in reversing a defect in transcription. These findings suggest that the drug sensitivity of elongation mutants is largely but not solely attributable to defects in the ability to induce IMD2, because transcription is compromised even when IMD2 mRNA levels are adequate. We describe two DNA sequence elements in the promoter of the gene that regulate it. We also found that IMD2 mRNA abundance is coupled to cell growth rate. These findings show that yeast possess a conserved system that gauges nucleotide pools and cell growth rate and responds through a uniquely regulated member of the IMD gene family.
George Weber - One of the best experts on this subject based on the ideXlab platform.
-
IMP Dehydrogenase inhibitor, tiazofurin, induces apoptosis in K562 human erythroleukemia cells
Cytometry, 1997Co-Authors: Marco Vitale, Loris Zamai, Elisabetta Falcieri, Giorgio Zauli, Pietro Gobbi, Spartaco Santi, Caterina Cinti, George WeberAbstract:Tiazofurin, an anticancer drug which inhibits IMP Dehydrogenase, decreases cellular GTP concentration, induces differentiation and down-regulates ras and myc oncogene expression, caused apoptosis of K562 cells in a time- and dose-dependent fashion. Apoptotic cells were detected by (1) flow cytometry, (2) electron microscopy, and (3) fluorescence in situ nick translation and confocal microscopy, while the DNA ladder was not detectable. The induced apoptosis was abrogated by guanosine which replenishes GTP pools through the guanosine salvage pathways, while it was enhanced by hypoxanthine, a competitive inhibitor of GPRT. The tiazofurin-mediated apoptosis may therefore be linked with the decrease of GTP and the consequent IMPairment of specific signal transduction pathways. Tiazofurin induced apoptosis also in lymphoblastic MOLT-4 cells, suggesting that this action is not confined to cells of the myeloid lineage, where the differentiating effects of the drug are more pronounced.
-
Proliferation-linked regulation of type II IMP Dehydrogenase gene in human normal lymphocytes and HL-60 leukemic cells
Cancer research, 1992Co-Authors: Masami Nagai, Yutaka Natsumeda, George WeberAbstract:Human IMP Dehydrogenase (IMPDH; EC 1.1.1.205) was recently found to consist of two molecular species (types I and II) with high expression of type II isozyme in leukemic cells. Here we report that the low level of type II mRNA in normal lymphocytes was up-regulated by phytohemagglutinin stimulation (3.2-fold) and Epstein-Barr viral transformation (5.7-fold). The type II mRNA expression in quiescent HL-60 cells was also elevated 2.8-fold by serum stimulation. Conversely the enhanced level of type II IMPDH mRNA in HL-60 cells was down-regulated to less than 5% along with differentiation induced by retinoic acid (1 microM), phorbol-12-myristate-13-acetate (33 nM), or dimethyl sulfoxide (1.25%) independent of end-stage phenotype. By contrast, type I IMPDH mRNA was expressed constitutively in the various states of proliferation and differentiation. The type II IMPDH stringently linked with cell proliferation should be a crucial target for antileukemic and immunosuppressive chemotherapy.
-
Reciprocal alterations of GMP reductase and IMP Dehydrogenase activities during differentiation in HL-60 leukemia cells.
Leukemia research, 1992Co-Authors: Hiroyuki Nakamura, Yutaka Natsumeda, Masami Nagai, Jiro Takahara, Shozo Irino, George WeberAbstract:The study was undertaken to elucidate the regulatory roles of GMP reductase (GMPR) and IMP Dehydrogenase (IMPDH) on purine interconversion during differentiation. Treatment of HL-60 cells with retinoic acid (1 microM) induced granulocytic differentiation which was accompanied with a 2.4-fold increase in GMPR and 55% decrease in IMPDH activities. Maturation induced by 12-O-tetradecanoylphorbol 13-acetate or dimethylsulfoxide was also associated with similar reciprocal alterations. Incubation with guanosine (200 microM), which expands the guanine nucleotide pool, elevated GMPR (1.9-fold) and decreased IMPDH (73%) activities. The synchronous and opposing alterations in GMPR and IMPDH activities should amplify the metabolic response due to differentiation or guanylate pool expansion.
-
Expression of human IMP Dehydrogenase types I and II in Escherichia coli and distribution in human normal lymphocytes and leukemic cell lines.
The Journal of biological chemistry, 1991Co-Authors: Y Konno, Shigeo Ohno, Yutaka Natsumeda, Yasufumi Yamaji, Masami Nagai, K Suzuki, George WeberAbstract:Two distinct cDNAs encoding proteins with 84% sequence identity have been isolated for human IMP Dehydrogenase (EC 1.1.1.205) (Natsumeda, Y., Ohno, S., Kawasaki, H., Konno, Y., Weber, G., and Suzuki, K. (1990) J. Biol. Chem. 265, 5292-5295), an IMPortant target in antileukemic chemotherapy. We constructed expression plasmids containing these cDNAs in full length with pUC plasmids and produced lacZ'-IMP Dehydrogenase fusion proteins in Escherichia coli. Both synthesized proteins exhibited IMP Dehydrogenase activity and were partially separated from endogenous E. coli IMP Dehydrogenase. By injecting the fusion proteins into mice we generated a polyclonal antibody specific to type I IMP Dehydrogenase and an antibody which reacted with both types. Immunoblot analysis revealed that the total amounts of types I and II enzymes increased in human leukemic cell lines K562 and HL-60 in agreement with the increase in IMP Dehydrogenase activity to 7.8- and 9.4-fold, respectively, above that of normal lymphocytes. The extent of expression of type I IMP Dehydrogenase was similar in these cells, however, indicating that the increase in IMP Dehydrogenase amount in leukemic cells was due to specific up-regulation of type II enzyme. Northern blot analysis also showed specific and predominant expression of type II in the leukemic cells. Thus, de novo GTP biosynthesis may be controlled differently in normal and neoplastic cells by different IMP Dehydrogenase molecular species.
-
IMP Dehydrogenase and GTP as targets in human leukemia treatment.
Advances in experimental medicine and biology, 1991Co-Authors: George WeberAbstract:GTP has multi-faceted cellular functions. Apart from its role in metabolism, in biosynthesis of RNA, proteins, biopterins, UTP and tubulin, GTP is an intricate part of signal transduction mechanisms, production of c-GMP and adenylates, G-protein action and expression of ras oncogene family. Guanylates are indispensable in DNA biosynthesis, since from GDP dGDP is formed and then dGTP, which is rate-limiting as it is the smallest pool among the dNTPs1,2 (Fig. 1). Curtailing GTP and dGTP pools is an IMPortant chemotherapeutic objective. GTP de novo biosynthesis is governed by IMP Dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme1,2. GTP pools are influenced by the activity of GPRT (guanine-hypoxanthine phosphoribosyltransferase, EC 2.4.2.8), the salvage enzyme, which can recycle guanine to GMP in one step. The significance of GTP in cancer biochemistry and chemotherapy was highlighted by the discovery that IMP Dehydrogenase activity increased in a transformation- and progression-linked fashion in rat hepatomas of different growth rates1,2. IMP Dehydrogenase activity increased in all murine and 4 human cancer cell lines and was particularly high in rapidly proliferating neoplastic cells such as leukemic cells1–3.
Yutaka Natsumeda - One of the best experts on this subject based on the ideXlab platform.
-
mycophenolic acid an inhibitor of IMP Dehydrogenase that is also an immunosuppressive agent suppresses the cytokine induced nitric oxide production in mouse and rat vascular endothelial cells
Transplantation, 1995Co-Authors: Mototaka Senda, Barbara Delustro, Elsie M Eugui, Yutaka NatsumedaAbstract:Mycophenolic acid (MPA), an inhibitor of IMP Dehydrogenase and de novo GTP biosynthesis, also has immunosuppressive activity. The effect of MPA on nitric oxide (NO) production by rodent brain vascular endothelial cells in culture was investigated. MPA inhibited NO production by mouse and rat brain endothelial cells that had been stimulated with a combination of interferon-gamma and tumor necrosis factor-alpha. The 50% inhibitory concentration (EC50) was in the range of 0.5-1.0 microM. However, MPA had no effect on basal NO production in mouse brain vascular endothelial cells. Brequinar, an inhibitor of de novo pyrimidine synthesis, had no effect on NO production in cytokine stimulated endothelial cells. Guanosine, which can act as a salvage pathway precursor for GTP biosynthesis, reversed the inhibitory effect of MPA in a dose-dependent fashion. We suggest that inducible NO synthase activity is dependent on GTP level and can be blocked by curtailing IMP Dehydrogenase activity.
-
Tissue-differential expression of two distinct genes for human IMP Dehydrogenase (E.C.1.1.1.205).
Life sciences, 1994Co-Authors: Mototaka Senda, Yutaka NatsumedaAbstract:Abstract Human IMP Dehydrogenase (E.C.1.1.205) is recently regarded as a potent targeting enzyme for immunosuppressive drugs. Tissue differential expressions of human type I and type II IMP Dehydrogenase were investigated in sixteen human adult organs (heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leukocytes) and five human fetal organs (heart, brain, lung, liver, kidney) using Northern blot analysis. In all tissues examined in this study, the sizes of mRNAs of each isoform were identical, respectively. The 2.3 kb type II mRNA was shown predominantly, and the 3.5 kb type I mRNA level was lower than type II in most human tissues examined. In contrast, type I IMPDH gene expressed higher than type II in peripheral blood leukocytes, uniquely. We also demonstrated that both type I and type II IMPDH genes are widely distributed among various species by Southern blot analysis. Interestingly, type I IMPDH gene may have multiple gene families in primates.
-
Proliferation-linked regulation of type II IMP Dehydrogenase gene in human normal lymphocytes and HL-60 leukemic cells
Cancer research, 1992Co-Authors: Masami Nagai, Yutaka Natsumeda, George WeberAbstract:Human IMP Dehydrogenase (IMPDH; EC 1.1.1.205) was recently found to consist of two molecular species (types I and II) with high expression of type II isozyme in leukemic cells. Here we report that the low level of type II mRNA in normal lymphocytes was up-regulated by phytohemagglutinin stimulation (3.2-fold) and Epstein-Barr viral transformation (5.7-fold). The type II mRNA expression in quiescent HL-60 cells was also elevated 2.8-fold by serum stimulation. Conversely the enhanced level of type II IMPDH mRNA in HL-60 cells was down-regulated to less than 5% along with differentiation induced by retinoic acid (1 microM), phorbol-12-myristate-13-acetate (33 nM), or dimethyl sulfoxide (1.25%) independent of end-stage phenotype. By contrast, type I IMPDH mRNA was expressed constitutively in the various states of proliferation and differentiation. The type II IMPDH stringently linked with cell proliferation should be a crucial target for antileukemic and immunosuppressive chemotherapy.
-
Reciprocal alterations of GMP reductase and IMP Dehydrogenase activities during differentiation in HL-60 leukemia cells.
Leukemia research, 1992Co-Authors: Hiroyuki Nakamura, Yutaka Natsumeda, Masami Nagai, Jiro Takahara, Shozo Irino, George WeberAbstract:The study was undertaken to elucidate the regulatory roles of GMP reductase (GMPR) and IMP Dehydrogenase (IMPDH) on purine interconversion during differentiation. Treatment of HL-60 cells with retinoic acid (1 microM) induced granulocytic differentiation which was accompanied with a 2.4-fold increase in GMPR and 55% decrease in IMPDH activities. Maturation induced by 12-O-tetradecanoylphorbol 13-acetate or dimethylsulfoxide was also associated with similar reciprocal alterations. Incubation with guanosine (200 microM), which expands the guanine nucleotide pool, elevated GMPR (1.9-fold) and decreased IMPDH (73%) activities. The synchronous and opposing alterations in GMPR and IMPDH activities should amplify the metabolic response due to differentiation or guanylate pool expansion.
-
Expression of human IMP Dehydrogenase types I and II in Escherichia coli and distribution in human normal lymphocytes and leukemic cell lines.
The Journal of biological chemistry, 1991Co-Authors: Y Konno, Shigeo Ohno, Yutaka Natsumeda, Yasufumi Yamaji, Masami Nagai, K Suzuki, George WeberAbstract:Two distinct cDNAs encoding proteins with 84% sequence identity have been isolated for human IMP Dehydrogenase (EC 1.1.1.205) (Natsumeda, Y., Ohno, S., Kawasaki, H., Konno, Y., Weber, G., and Suzuki, K. (1990) J. Biol. Chem. 265, 5292-5295), an IMPortant target in antileukemic chemotherapy. We constructed expression plasmids containing these cDNAs in full length with pUC plasmids and produced lacZ'-IMP Dehydrogenase fusion proteins in Escherichia coli. Both synthesized proteins exhibited IMP Dehydrogenase activity and were partially separated from endogenous E. coli IMP Dehydrogenase. By injecting the fusion proteins into mice we generated a polyclonal antibody specific to type I IMP Dehydrogenase and an antibody which reacted with both types. Immunoblot analysis revealed that the total amounts of types I and II enzymes increased in human leukemic cell lines K562 and HL-60 in agreement with the increase in IMP Dehydrogenase activity to 7.8- and 9.4-fold, respectively, above that of normal lymphocytes. The extent of expression of type I IMP Dehydrogenase was similar in these cells, however, indicating that the increase in IMP Dehydrogenase amount in leukemic cells was due to specific up-regulation of type II enzyme. Northern blot analysis also showed specific and predominant expression of type II in the leukemic cells. Thus, de novo GTP biosynthesis may be controlled differently in normal and neoplastic cells by different IMP Dehydrogenase molecular species.
