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Mitchell, E. P., L. N. Johnson, Ph Ermert, A. T. Vasella, S. G. Withers, and N. G. Oikonomakos. "Crystallographic studies towards the catalytic mechanism of glycogen phosphorylase." Acta Crystallographica Section A Foundations of Crystallography 49, s1 (August 21, 1993): c87. http://dx.doi.org/10.1107/s0108767378097512.
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Papageorgiou, A. C., N. G. Oikonomakos, D. D. Leonidas, B. Bernet, D. Beer, and A. Vasella. "The binding of d-gluconohydroximo-1,5-lactone to glycogen phosphorylase. Kinetic, ultracentrifugation and crystallographic studies." Biochemical Journal 274, no. 2 (March 1, 1991): 329–38. http://dx.doi.org/10.1042/bj2740329.
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Combined kinetic, ultracentrifugation and X-ray-crystallographic studies have characterized the effect of the beta-glucosidase inhibitor gluconohydroximo-1,5-lactone on the catalytic and structural properties of glycogen phosphorylase. In the direction of glycogen synthesis, gluconohydroximo-1,5-lactone was found to competitively inhibit both the b (Ki 0.92 mM) and the alpha form of the enzyme (Ki 0.76 mM) with respect to glucose 1-phosphate in synergism with caffeine. In the direction of glycogen breakdown, gluconohydroximo-1,5-lactone was found to inhibit phosphorylase b in a non-competitive mode with respect to phosphate, and no synergism with caffeine could be demonstrated. Ultracentrifugation and crystallization experiments demonstrated that gluconohydroximo-1,5-lactone was able to induce dissociation of tetrameric phosphorylase alpha and stabilization of the dimeric T-state conformation. A crystallographic binding study with 100 mM-gluconohydroximo-1,5-lactone at 0.24 nm (2.4 A) resolution showed a major peak at the catalytic site, and no significant conformational changes were observed. Analysis of the electron-density map indicated that the ligand adopts a chair conformation. The results are discussed with reference to the ability of the catalytic site of the enzyme to distinguish between two or more conformations of the glucopyranose ring.
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Mavreas, Konstantinos F., Dionysios D. Neofytos, Evangelia D. Chrysina, Alessandro Venturini, and Thanasis Gimisis. "Synthesis, Kinetic and Conformational Studies of 2-Substituted-5-(β-d-glucopyranosyl)-pyrimidin-4-ones as Potential Inhibitors of Glycogen Phosphorylase." Molecules 25, no. 22 (November 22, 2020): 5463. http://dx.doi.org/10.3390/molecules25225463.
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Dysregulation of glycogen phosphorylase, an enzyme involved in glucose homeostasis, may lead to a number of pathological states such as type 2 diabetes and cancer, making it an important molecular target for the development of new forms of pharmaceutical intervention. Based on our previous work on the design and synthesis of 4-arylamino-1-(β-d-glucopyranosyl)pyrimidin-2-ones, which inhibit the activity of glycogen phosphorylase by binding at its catalytic site, we report herein a general synthesis of 2-substituted-5-(β-d-glucopyranosyl)pyrimidin-4-ones, a related class of metabolically stable, C-glucosyl-based, analogues. The synthetic development consists of a metallated heterocycle, produced from 5-bromo-2-methylthiouracil, in addition to protected d-gluconolactone, followed by organosilane reduction. The methylthio handle allowed derivatization through hydrolysis, ammonolysis and arylamine substitution, and the new compounds were found to be potent (μM) inhibitors of rabbit muscle glycogen phosphorylase. The results were interpreted with the help of density functional theory calculations and conformational analysis and were compared with previous findings.
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Gruppuso, P. A., and D. L. Brautigan. "Induction of hepatic glycogenesis in the fetal rat." American Journal of Physiology-Endocrinology and Metabolism 256, no. 1 (January 1, 1989): E49—E54. http://dx.doi.org/10.1152/ajpendo.1989.256.1.e49.
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We have performed an in vivo study to test the hypothesis that induction of fetal hepatic glycogenesis is stimulated by insulin and involves activation of protein phosphatase type-1. Control animals and the following two experimental groups were studied: maternal fasting for 48 h prior to term and chronic maternal hyperinsulinemia for 5 days prior to term. Maternal fasting led to decreased fetal hepatic glycogen content and fetal growth retardation. In contrast, no decrease in fetal hepatic glycogen content or fetal weight occurred with maternal hyperinsulinemia despite fetal hypoglycemia and fetal hypoinsulinemia. In neither model were fetal hepatic synthase phosphatase or phosphorylase phosphatase activities affected. In control fetuses, the appearance of hepatic glycogen from days 17 to 21 of gestation correlated with induction of glycogen synthase. Phosphorylase phosphatase and synthase phosphatase activities already were present on day 17 of gestation and changed little through term. However, phosphatase catalytic protein reactive with anti-phosphatase type-1 antibodies did increase approximately fivefold from day 18 to 21. In adult animals fasted for 48 h, 50% of hepatic glycogen synthase phosphatase activity was lost, whereas phosphorylase phosphatase activity was stimulated fourfold. The apparent size of protein phosphatase type-1 catalytic subunit as detected by Western immunoblotting was altered by fasting in the adult but not by substrate restriction (maternal fasting) in the fetus.(ABSTRACT TRUNCATED AT 250 WORDS)
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Acharya, K. R., J. Hajdu, D. I. Stuart, P. J. McLaughlin, D. Barford, N. G. Oikonomakos, and L. N. Johnson. "Catalysis in the crystal: synchrotron radiation studies with glycogen phosphorylaseb." Acta Crystallographica Section A Foundations of Crystallography 43, a1 (August 12, 1987): C40. http://dx.doi.org/10.1107/s0108767387084393.
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Tu, J., and M. Carlson. "The GLC7 type 1 protein phosphatase is required for glucose repression in Saccharomyces cerevisiae." Molecular and Cellular Biology 14, no. 10 (October 1994): 6789–96. http://dx.doi.org/10.1128/mcb.14.10.6789.
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We cloned the GLC7/DIS2S1 gene by complementation of the cid1-226 mutation, which relieves glucose repression in Saccharomyces cerevisiae. GLC7 encodes the catalytic subunit of type 1 protein phosphatase (PP1). Genetic analysis and sequencing showed that cid1-226 is an allele of GLC7, now designated glc7-T152K, which alters threonine 152 to lysine. We also show that the glc7-1 and glc7-T152K alleles cause distinct phenotypes: glc7-1 causes a severe defect in glycogen accumulation but does not relieve glucose repression, whereas glc7-T152K does not prevent glycogen accumulation. These findings are discussed in light of evidence that interaction with different regulatory or targeting subunits directs the participation of PP1 in diverse cellular regulatory mechanisms. Finally, genetic studies suggest that PP1 functions antagonistically to the SNF1 protein kinase in the regulatory response to glucose.
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Tu, J., and M. Carlson. "The GLC7 type 1 protein phosphatase is required for glucose repression in Saccharomyces cerevisiae." Molecular and Cellular Biology 14, no. 10 (October 1994): 6789–96. http://dx.doi.org/10.1128/mcb.14.10.6789-6796.1994.
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We cloned the GLC7/DIS2S1 gene by complementation of the cid1-226 mutation, which relieves glucose repression in Saccharomyces cerevisiae. GLC7 encodes the catalytic subunit of type 1 protein phosphatase (PP1). Genetic analysis and sequencing showed that cid1-226 is an allele of GLC7, now designated glc7-T152K, which alters threonine 152 to lysine. We also show that the glc7-1 and glc7-T152K alleles cause distinct phenotypes: glc7-1 causes a severe defect in glycogen accumulation but does not relieve glucose repression, whereas glc7-T152K does not prevent glycogen accumulation. These findings are discussed in light of evidence that interaction with different regulatory or targeting subunits directs the participation of PP1 in diverse cellular regulatory mechanisms. Finally, genetic studies suggest that PP1 functions antagonistically to the SNF1 protein kinase in the regulatory response to glucose.
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Hajdu, J., K. R. Acharya, D. I. Stuart, P. J. McLaughlin, D. Barford, N. G. Oikonomakos, H. Klein, and L. N. Johnson. "Catalysis in the crystal: synchrotron radiation studies with glycogen phosphorylase b." EMBO Journal 6, no. 2 (February 1987): 539–46. http://dx.doi.org/10.1002/j.1460-2075.1987.tb04786.x.
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Coate, Katie C., Guillaume Kraft, Masakazu Shiota, Marta S. Smith, Ben Farmer, Doss W. Neal, Phil Williams, Alan D. Cherrington, and Mary Courtney Moore. "Chronic overeating impairs hepatic glucose uptake and disposition." American Journal of Physiology-Endocrinology and Metabolism 308, no. 10 (May 15, 2015): E860—E867. http://dx.doi.org/10.1152/ajpendo.00069.2015.
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Dogs consuming a hypercaloric high-fat and -fructose diet (52 and 17% of total energy, respectively) or a diet high in either fructose or fat for 4 wk exhibited blunted net hepatic glucose uptake (NHGU) and glycogen deposition in response to hyperinsulinemia, hyperglycemia, and portal glucose delivery. The effect of a hypercaloric diet containing neither fructose nor excessive fat has not been examined. Dogs with an initial weight of ≈25 kg consumed a chow and meat diet (31% protein, 44% carbohydrate, and 26% fat) in weight-maintaining (CTR; n = 6) or excessive (Hkcal; n = 7) amounts for 4 wk (cumulative weight gain 0.0 ± 0.3 and 1.5 ± 0.5 kg, respectively, P < 0.05). They then underwent clamp studies with infusions of somatostatin and intraportal insulin (4× basal) and glucagon (basal). The hepatic glucose load was doubled with peripheral (Pe) glucose infusion for 90 min (P1) and intraportal glucose at 4 mg·kg−1·min−1 plus Pe glucose for the final 90 min (P2). NHGU was blunted ( P < 0.05) in Hkcal during both periods (mg·kg−1·min−1; P1: 1.7 ± 0.2 vs. 0.3 ± 0.4; P2: 3.6 ± 0.3 vs. 2.3 ± 0.4, CTR vs. Hkcal, respectively). Terminal hepatic glucokinase catalytic activity was reduced nearly 50% in Hkcal vs. CTR ( P < 0.05), although glucokinase protein did not differ between groups. In Hkcal vs. CTR, liver glycogen was reduced 27% ( P < 0.05), with a 91% increase in glycogen phosphorylase activity ( P < 0.05) but no significant difference in glycogen synthase activity. Thus, Hkcal impaired NHGU and glycogen synthesis compared with CTR, indicating that excessive energy intake, even if the diet is balanced and nutritious, negatively impacts hepatic glucose metabolism.
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Zographos, S. E., N. G. Oikonomakos, H. B. F. Dixon, W. G. Griffin, L. N. Johnson, and D. D. Leonidas. "Sulphate-activated phosphorylase b: the pH-dependence of catalytic activity." Biochemical Journal 310, no. 2 (September 1, 1995): 565–70. http://dx.doi.org/10.1042/bj3100565.
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The pH-dependence of sulphate-activated phosphorylase b has been studied in the direction of glycogen synthesis. The bell-shaped curve of the pH-dependence of the catalytic constant for the AMP-activated enzyme showed pK values of 6.1 and 7.3, but the curve for the enzyme activated by 0.9 M ammonium sulphate showed a drop of activity on the acid side at much higher pH values. Its bell was centred at pH 7.8 but it was too narrow to be characterized by only two pK values. The narrowness of the curve could be explained by positive co-operativity, but not its unusually steep acid side. We suggest that the fall on the acid side is due to more than one hydronation (addition of H+). The points can be fitted by a curve with two de-activating hydronations and a de-activating dehydronation having identical titration pK values of 7.5, and hence molecular values of 7.0, 7.5 and 8.0. If both 0.9 M ammonium sulphate and 5 mM AMP are added, the bell is as broad as with AMP alone, but is somewhat raised in pH optimum. The results are discussed in the light of new structural data from crystallographic studies on binary complexes of the enzyme.
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Varadharaj, Vanitha, and Naresh Kandakatl. "GLYCOGEN SYNTHASE KINASE-3 BETA PROTEIN INHIBITION BY SELECTED PHYTOCOMPOUNDS IN SILICO." Asian Journal of Pharmaceutical and Clinical Research 10, no. 1 (January 1, 2016): 87. http://dx.doi.org/10.22159/ajpcr.2017.v10i1.14113.
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ABSTRACTObjectives: Bioactive phytocompounds are a rich source of chemopreventive substance. In the present investigation, docking study was performedfor the selected bioactive phytocompounds such as oleanolic acid, ecdysterone, betaine, stigmasterol acetate, and cinnamic acid to evaluate theiraffinity to glycogen synthase kinase-3 beta (GSK-3β) protein, a wound-healing biomarker. 2-chloro-5-[4-(3-chloro-phenyl)-2, 5-dioxo-2,5-dihydro-1hpyrrol-3-ylamino]-benzoicacid wasused as an inhibitorforGSK-3βwith minimum binding energy(−31.5 kcal/mol).Methods: Molecular docking study was conducted using AutoDock 4.2 version and the visualization result using Discover Studio 4.5.Results: The docking analysis ranked the selected phytocompounds that have high theoretical scores to bind to the proteins. The binding mode of thephytocompounds that bound to all the target proteins with high affinity was studied. The simulation demonstrated that the protein-ligand complexstabilized by multiple hydrogen bonds (H-bonds) was preferentially formed at the catalytic site. The results highlighted in this study reveals thatamong the selected lead phytocompounds that docked into the active site of GSK-3β, ecdysterone showed acceptable 6 H-bond interactions withresidues LYS85, TYR134, ARG141, GLN185, ASP200, PRO136 when compared to the reference compound with 5 H-bond interactions.Conclusion: Thus, based on the docking score ecdysterone could be considered as a novel compounds that can be used for experimental studies forthe inhibition of GSK-3β kinase. These results can be helpful for further design of novel GSK-3β inhibitors.Keywords: Phytocompounds, Molecular docking, Simulation, Receptor, Ligand, Inhibition.
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HAJDU, JANOS, K. RAVI ACHARYA, DAVID I. STUART, PAUL J. McLAUGHLIN, DAVID BARFORD, HELMUT KLEIN, and LOUISE N. JOHNSON. "Time-resolved structural studies on catalysis in the crystal with glycogen phosphorylase b." Biochemical Society Transactions 14, no. 3 (June 1, 1986): 538–41. http://dx.doi.org/10.1042/bst0140538.
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Rahimpour, Mehdi, Manuel Montero, Goizeder Almagro, Alejandro M. Viale, Ángel Sevilla, Manuel Cánovas, Francisco J. Muñoz, et al. "GlgS, described previously as a glycogen synthesis control protein, negatively regulates motility and biofilm formation in Escherichia coli." Biochemical Journal 452, no. 3 (May 31, 2013): 559–73. http://dx.doi.org/10.1042/bj20130154.
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Escherichia coli glycogen metabolism involves the regulation of glgBXCAP operon expression and allosteric control of the GlgC [ADPG (ADP-glucose) pyrophosphorylase]-mediated catalysis of ATP and G1P (glucose-1-phosphate) to ADPG linked to glycogen biosynthesis. E. coli glycogen metabolism is also affected by glgS. Though the precise function of the protein it encodes is unknown, its deficiency causes both reduced glycogen content and enhanced levels of the GlgC-negative allosteric regulator AMP. The transcriptomic analyses carried out in the present study revealed that, compared with their isogenic BW25113 wild-type strain, glgS-null (ΔglgS) mutants have increased expression of the operons involved in the synthesis of type 1 fimbriae adhesins, flagella and nucleotides. In agreement, ΔglgS cells were hyperflagellated and hyperfimbriated, and displayed elevated swarming motility; these phenotypes all reverted to the wild-type by ectopic glgS expression. Also, ΔglgS cells accumulated high colanic acid content and displayed increased ability to form biofilms on polystyrene surfaces. F-driven conjugation based on large-scale interaction studies of glgS with all the non-essential genes of E. coli showed that deletion of purine biosynthesis genes complement the glycogen-deficient, high motility and high biofilm content phenotypes of ΔglgS cells. Overall the results of the present study indicate that glycogen deficiency in ΔglgS cells can be ascribed to high flagellar propulsion and high exopolysaccharide and purine nucleotides biosynthetic activities competing with GlgC for the same ATP and G1P pools. Supporting this proposal, glycogen-less ΔglgC cells displayed an elevated swarming motility, and accumulated high levels of colanic acid and biofilm. Furthermore, glgC overexpression reverted the glycogen-deficient, high swarming motility, high colanic acid and high biofilm content phenotypes of ΔglgS cells to the wild-type. As on the basis of the present study GlgS has emerged as a major determinant of E. coli surface composition and because its effect on glycogen metabolism appears to be only indirect, we propose to rename it as ScoR (surface composition regulator).
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Coate, Katie C., Guillaume Kraft, Mary Courtney Moore, Marta S. Smith, Christopher Ramnanan, Jose M. Irimia, Peter J. Roach, et al. "Hepatic glucose uptake and disposition during short-term high-fat vs. high-fructose feeding." American Journal of Physiology-Endocrinology and Metabolism 307, no. 2 (July 15, 2014): E151—E160. http://dx.doi.org/10.1152/ajpendo.00083.2014.
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In dogs consuming a high-fat and -fructose diet (52 and 17% of total energy, respectively) for 4 wk, hepatic glucose uptake (HGU) in response to hyperinsulinemia, hyperglycemia, and portal glucose delivery is markedly blunted with reduction in glucokinase (GK) protein and glycogen synthase (GS) activity. The present study compared the impact of selective increases in dietary fat and fructose on liver glucose metabolism. Dogs consumed weight-maintaining chow (CTR) or hypercaloric high-fat (HFA) or high-fructose (HFR) diets diet for 4 wk before undergoing clamp studies with infusion of somatostatin and intraportal insulin (3–4 times basal) and glucagon (basal). The hepatic glucose load (HGL) was doubled during the clamp using peripheral vein (Pe) glucose infusion in the first 90 min (P1) and portal vein (4 mg·kg−1·min−1) plus Pe glucose infusion during the final 90 min (P2). During P2, HGU was 2.8 ± 0.2, 1.0 ± 0.2, and 0.8 ± 0.2 mg·kg−1·min−1 in CTR, HFA, and HFR, respectively ( P < 0.05 for HFA and HFR vs. CTR). Compared with CTR, hepatic GK protein and catalytic activity were reduced ( P < 0.05) 35 and 56%, respectively, in HFA, and 53 and 74%, respectively, in HFR. Liver glycogen concentrations were 20 and 38% lower in HFA and HFR than CTR ( P < 0.05). Hepatic Akt phosphorylation was decreased ( P < 0.05) in HFA (21%) but not HFR. Thus, HFR impaired hepatic GK and glycogen more than HFA, whereas HFA reduced insulin signaling more than HFR. HFA and HFR effects were not additive, suggesting that they act via the same mechanism or their effects converge at a saturable step.
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Gibbons, Jennifer A., Douglas C. Weiser, and Shirish Shenolikar. "Importance of a Surface Hydrophobic Pocket on Protein Phosphatase-1 Catalytic Subunit in Recognizing Cellular Regulators." Journal of Biological Chemistry 280, no. 16 (February 9, 2005): 15903–11. http://dx.doi.org/10.1074/jbc.m500871200.
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Cellular functions of protein phosphatase-1 (PP1), a major eukaryotic serine/threonine phosphatase, are defined by the association of PP1 catalytic subunits with endogenous protein inhibitors and regulatory subunits. Many PP1 regulators share a consensus RVXF motif, which docks within a hydrophobic pocket on the surface of the PP1 catalytic subunit. Although these regulatory proteins also possess additional PP1-binding sites, mutations of the RVXF sequence established a key role of this PP1-binding sequence in the function of PP1 regulators. WT PP1α, the C-terminal truncated PP1α-(1–306), a chimeric PP1α containing C-terminal sequences from PP2A, another phosphatase, PP1α-(1–306) with the RVXF-binding pocket substitutions L289R, M290K, and C291R, and PP2A were analyzed for their regulation by several mammalian proteins. These studies established that modifications of the RVXF-binding pocket had modest effects on the catalytic activity of PP1, as judged by recognition of substrates and sensitivity to toxins. However, the selected modifications impaired the sensitivity of PP1 to the inhibitor proteins, inhibitor-1 and inhibitor-2. In addition, they impaired the ability of PP1 to bind neurabin-I, the neuronal regulatory subunit, and GM, the skeletal muscle glycogen-targeting subunit. These data suggested that differences in RVXF interactions with the hydrophobic pocket dictate the affinity of PP1 for cellular regulators. Substitution of a distinct RVXF sequence in inhibitor-1 that enhanced its binding and potency as a PP1 inhibitor emphasized the importance of the RVXF sequence in defining the function of this and other PP1 regulators. Our studies suggest that the diversity of RVXF sequences provides for dynamic physiological regulation of PP1 functions in eukaryotic cells.
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Lorenzo-Morales, Jacob, Carmen M. Martín-Navarro, Atteneri López-Arencibia, María A. Santana-Morales, Raquel N. Afonso-Lehmann, Sutherland K. Maciver, Basilio Valladares, and Enrique Martínez-Carretero. "Therapeutic Potential of a Combination of Two Gene-Specific Small Interfering RNAs against Clinical Strains of Acanthamoeba." Antimicrobial Agents and Chemotherapy 54, no. 12 (September 20, 2010): 5151–55. http://dx.doi.org/10.1128/aac.00329-10.
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ABSTRACT Pathogenic strains of the genus Acanthamoeba are causative agents of severe infections, such as fatal encephalitis and a sight-threatening amoebic keratitis. Antimicrobial therapy for these infections is generally empirical, and patient recovery is often problematic, due to the existence of a highly resistant cyst stage in these amoebae. In previous studies, small interfering RNAs (siRNAs) against the catalytic domains of extracellular serine proteases and glycogen phosphorylase from Acanthamoeba were designed and evaluated for future therapeutic use. The silencing of proteases resulted in Acanthamoeba failing to degrade human corneal cells, and silencing of glycogen phosphorylase caused amoebae to be unable to form mature cysts. After the siRNA design and concentration were optimized in order to avoid toxicity problems, cultures of Acanthamoeba were treated with a combination of both siRNAs, and cells were evaluated under an inverted microscope. This siRNA-based treatment dramatically affected the growth rate and cellular survival of the amoebae. These results were observed less than 48 h after the initiation of the treatment. In order to check possible toxic effects of the siRNA combination, three eukaryotic cell lines (HeLa, murine macrophages, and osteosarcoma cells) were treated with the same molecules, and cytotoxicity was examined by measuring lactate dehydrogenase release. The future use of the combination of these siRNAs is proposed as a potential therapeutic approach against pathogenic strains of Acanthamoeba.
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Chrysina, E. D., N. G. Oikonomakos, S. E. Zographos, M. N. Kosmopoulou, N. Bischler, D. D. Leonidas, L. Kovács, T. Docsa, P. Gergely, and L. Somsák. "Crystallographic Studies on α- and β-D-glucopyranosyl Formamide Analogues, Inhibitors of Glycogen Phosphorylase." Biocatalysis and Biotransformation 21, no. 4-5 (October 2003): 233–42. http://dx.doi.org/10.1080/10242420310001614360.
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Heightman, Tom D., Andrea Vasella, Katerina E. Tsitsanou, Spyros E. Zographos, Vicky T. Skamnaki, and Nikos G. Oikonomakos. "Cooperative Interactions of the Catalytic Nucleophile and the Catalytic Acid in the Inhibition of ?-Glycosidases. Calculations and their validation by comparative kinetic and structural studies of the inhibition of glycogen phosphorylaseb." Helvetica Chimica Acta 81, no. 5-8 (1998): 853–64. http://dx.doi.org/10.1002/hlca.19980810507.
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Barford, D., J. W. R. Schwabe, N. G. Oikonomakos, K. R. Acharya, J. Hajdu, A. C. Papageorgiou, J. L. Martin, J. C. A. Knott, A. Vasella, and L. N. Johnson. "Channels at the catalytic site of glycogen phosphorylase b: binding and kinetic studies with the .beta.-glycosidase inhibitor D-gluconohydroximo-1,5-lactone N-phenylurethane." Biochemistry 27, no. 18 (September 6, 1988): 6733–41. http://dx.doi.org/10.1021/bi00418a014.
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Leoni, Claudia, Bruno A. R. Gattulli, Graziano Pesole, Luigi R. Ceci, and Mariateresa Volpicella. "Amylomaltases in Extremophilic Microorganisms." Biomolecules 11, no. 9 (September 9, 2021): 1335. http://dx.doi.org/10.3390/biom11091335.
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Amylomaltases (4-α-glucanotransferases, E.C. 2.4.1.25) are enzymes which can perform a double-step catalytic process, resulting in a transglycosylation reaction. They hydrolyse glucosidic bonds of α-1,4′-d-glucans and transfer the glucan portion with the newly available anomeric carbon to the 4′-position of an α-1,4′-d-glucan acceptor. The intramolecular reaction produces a cyclic α-1,4′-glucan. Amylomaltases can be found only in prokaryotes, where they are involved in glycogen degradation and maltose metabolism. These enzymes are being studied for possible biotechnological applications, such as the production of (i) sugar substitutes; (ii) cycloamyloses (molecules larger than cyclodextrins), which could potentially be useful as carriers and encapsulating agents for hydrophobic molecules and also as effective protein chaperons; and (iii) thermoreversible starch gels, which could be used as non-animal gelatin substitutes. Extremophilic prokaryotes have been investigated for the identification of amylomaltases to be used in the starch modifying processes, which require high temperatures or extreme conditions. The aim of this article is to present an updated overview of studies on amylomaltases from extremophilic Bacteria and Archaea, including data about their distribution, activity, potential industrial application and structure.
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Ballicora, Miguel A., Esteban D. Erben, Terutaka Yazaki, Ana L. Bertolo, Ana M. Demonte, Jennifer R. Schmidt, Mabel Aleanzi, et al. "Identification of Regions Critically Affecting Kinetics and Allosteric Regulation of the Escherichia coli ADP-Glucose Pyrophosphorylase by Modeling and Pentapeptide-Scanning Mutagenesis." Journal of Bacteriology 189, no. 14 (May 11, 2007): 5325–33. http://dx.doi.org/10.1128/jb.00481-07.
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ABSTRACT ADP-glucose pyrophosphorylase (ADP-Glc PPase) is the enzyme responsible for the regulation of bacterial glycogen synthesis. To perform a structure-function relationship study of the Escherichia coli ADP-Glc PPase enzyme, we studied the effects of pentapeptide insertions at different positions in the enzyme and analyzed the results with a homology model. We randomly inserted 15 bp in a plasmid with the ADP-Glc PPase gene. We obtained 140 modified plasmids with single insertions of which 21 were in the coding region of the enzyme. Fourteen of them generated insertions of five amino acids, whereas the other seven created a stop codon and produced truncations. Correlation of ADP-Glc PPase activity to these modifications validated the enzyme model. Six of the insertions and one truncation produced enzymes with sufficient activity for the E. coli cells to synthesize glycogen and stain in the presence of iodine vapor. These were in regions away from the substrate site, whereas the mutants that did not stain had alterations in critical areas of the protein. The enzyme with a pentapeptide insertion between Leu102 and Pro103 was catalytically competent but insensitive to activation. We postulate this region as critical for the allosteric regulation of the enzyme, participating in the communication between the catalytic and regulatory domains.
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Leiba, Jade, Karl Syson, Grégory Baronian, Isabelle Zanella-Cléon, Rainer Kalscheuer, Laurent Kremer, Stephen Bornemann, and Virginie Molle. "Mycobacterium tuberculosis Maltosyltransferase GlgE, a Genetically Validated Antituberculosis Target, Is Negatively Regulated by Ser/Thr Phosphorylation." Journal of Biological Chemistry 288, no. 23 (April 22, 2013): 16546–56. http://dx.doi.org/10.1074/jbc.m112.398503.
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GlgE is a maltosyltransferase involved in the biosynthesis of α-glucans that has been genetically validated as a potential therapeutic target against Mycobacterium tuberculosis. Despite also making α-glucan, the GlgC/GlgA glycogen pathway is distinct and allosterically regulated. We have used a combination of genetics and biochemistry to establish how the GlgE pathway is regulated. M. tuberculosis GlgE was phosphorylated specifically by the Ser/Thr protein kinase PknB in vitro on one serine and six threonine residues. Furthermore, GlgE was phosphorylated in vivo when expressed in Mycobacterium bovis bacillus Calmette–Guérin (BCG) but not when all seven phosphorylation sites were replaced by Ala residues. The GlgE orthologues from Mycobacterium smegmatis and Streptomyces coelicolor were phosphorylated by the corresponding PknB orthologues in vitro, implying that the phosphorylation of GlgE is widespread among actinomycetes. PknB-dependent phosphorylation of GlgE led to a 2 orders of magnitude reduction in catalytic efficiency in vitro. The activities of phosphoablative and phosphomimetic GlgE derivatives, where each phosphorylation site was substituted with either Ala or Asp residues, respectively, correlated with negative phosphoregulation. Complementation studies of a M. smegmatis glgE mutant strain with these GlgE derivatives, together with both classical and chemical forward genetics, were consistent with flux through the GlgE pathway being correlated with GlgE activity. We conclude that the GlgE pathway appears to be negatively regulated in actinomycetes through the phosphorylation of GlgE by PknB, a mechanism distinct from that known in the classical glycogen pathway. Thus, these findings open new opportunities to target the GlgE pathway therapeutically.
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Ballicora, Miguel A., Alberto A. Iglesias, and Jack Preiss. "ADP-Glucose Pyrophosphorylase, a Regulatory Enzyme for Bacterial Glycogen Synthesis." Microbiology and Molecular Biology Reviews 67, no. 2 (June 2003): 213–25. http://dx.doi.org/10.1128/mmbr.67.2.213-225.2003.
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SUMMARY The accumulation of α-1,4-polyglucans is an important strategy to cope with transient starvation conditions in the environment. In bacteria and plants, the synthesis of glycogen and starch occurs by utilizing ADP-glucose as the glucosyl donor for elongation of the α-1,4-glucosidic chain. The main regulatory step takes place at the level of ADP-glucose synthesis, a reaction catalyzed by ADP-Glc pyrophosphorylase (PPase). Most of the ADP-Glc PPases are allosterically regulated by intermediates of the major carbon assimilatory pathway in the organism. Based on specificity for activator and inhibitor, classification of ADP-Glc PPases has been expanded into nine distinctive classes. According to predictions of the secondary structure of the ADP-Glc PPases, they seem to have a folding pattern common to other sugar nucleotide pyrophosphorylases. All the ADP-Glc PPases as well as other sugar nucleotide pyrophosphorylases appear to have evolved from a common ancestor, and later, ADP-Glc PPases developed specific regulatory properties, probably by addition of extra domains. Studies of different domains by construction of chimeric ADP-Glc PPases support this hypothesis. In addition to previous chemical modification experiments, the latest random and site-directed mutagenesis experiments with conserved amino acids revealed residues important for catalysis and regulation.
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Molares-Vila, Alberto, Alberte Corbalán-Rivas, Miguel Carnero-Gregorio, José Luís González-Cespón, and Carmen Rodríguez-Cerdeira. "Biomarkers in Glycogen Storage Diseases: An Update." International Journal of Molecular Sciences 22, no. 9 (April 22, 2021): 4381. http://dx.doi.org/10.3390/ijms22094381.
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Glycogen storage diseases (GSDs) are a group of 19 hereditary diseases caused by a lack of one or more enzymes involved in the synthesis or degradation of glycogen and are characterized by deposits or abnormal types of glycogen in tissues. Their frequency is very low and they are considered rare diseases. Except for X-linked type IX, the different types are inherited in an autosomal recessive pattern. In this study we reviewed the literature from 1977 to 2020 concerning GSDs, biomarkers, and metabolic imbalances in the symptoms of some GSDs. Most of the reported studies were performed with very few patients. Classification of emerging biomarkers between different types of diseases (hepatics GSDs, McArdle and PDs and other possible biomarkers) was done for better understanding. Calprotectin for hepatics GSDs and urinary glucose tetrasaccharide for Pompe disease have been approved for clinical use, and most of the markers mentioned in this review only need clinical validation, as a final step for their routine use. Most of the possible biomarkers are implied in hepatocellular adenomas, cardiomyopathies, in malfunction of skeletal muscle, in growth retardation, neutropenia, osteopenia and bowel inflammation. However, a few markers have lost interest due to a great variability of results, which is the case of biotinidase, actin alpha 2, smooth muscle, aorta and fibroblast growth factor receptor 4. This is the first review published on emerging biomarkers with a potential application to GSDs.
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Santos, M., S. Rebelo, O. A. B. da Cruz e. Silva, and E. F. da Cruz e. Silva. "Immunolocalization of PPP1C Isoforms in SH-SY5Y Cells During the Cell Cycle." Microscopy and Microanalysis 18, S5 (August 2012): 41–42. http://dx.doi.org/10.1017/s143192761201286x.
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Phosphoprotein phosphatase 1 (PPP1) is the most widely expressed and abundant serine/threonine protein phosphatase. PPP1 regulates a variety of cellular functions such as glycogen metabolism, mitosis and meiosis, cell-cycle arrest, apoptosis, dynamics of actin cytoskeleton, protein synthesis among others. The versatility of the PPP1 catalytic subunit (PPP1C) is achieved by associating with different regulatory subunits that target PPP1C to a particular subcellular compartment determining its substrate specificity and activity. PPP1C is expressed in mammals in three isoforms: PPP1CA, PPP1CB and PPP1CC, each encoded by distinct genes. The PPP1C gene undergoes alternative splicing to originate PPP1CC1 and PPP1CC2 variants. PPP1A and PPP1C1 are expressed virtually in all tissues but are particularly enriched in the brain. The subcellular localization of the endogenous PPP1C isoforms is not fully elucidated, but all isoforms are found in the nucleus and cytoplasm, despite some isoform-specific differences in intranuclear distribution. Furthermore, it is known that PPP1 plays a key role in mitosis where PPP1C isoforms are differentially targeted to specific subcellular structures. However, in previous studies, non-neuronal cells were used as a model system to study PPP1 distribution during the cell cycle. In the studies here described, we used the neuronal-like cell line SH-SY5Y since PPP1 is a crucial protein in several neuronal functions.
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Llavero, Arrazola Sastre, Luque Montoro, Gálvez, Lacerda, Parada, and Zugaza. "McArdle Disease: New Insights into Its Underlying Molecular Mechanisms." International Journal of Molecular Sciences 20, no. 23 (November 25, 2019): 5919. http://dx.doi.org/10.3390/ijms20235919.
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McArdle disease, also known as glycogen storage disease type V (GSDV), is characterized by exercise intolerance, the second wind phenomenon, and high serum creatine kinase activity. Here, we recapitulate PYGM mutations in the population responsible for this disease. Traditionally, McArdle disease has been considered a metabolic myopathy caused by the lack of expression of the muscle isoform of the glycogen phosphorylase (PYGM). However, recent findings challenge this view, since it has been shown that PYGM is present in other tissues than the skeletal muscle. We review the latest studies about the molecular mechanism involved in glycogen phosphorylase activity regulation. Further, we summarize the expression and functional significance of PYGM in other tissues than skeletal muscle both in health and McArdle disease. Furthermore, we examine the different animal models that have served as the knowledge base for better understanding of McArdle disease. Finally, we give an overview of the latest state-of-the-art clinical trials currently being carried out and present an updated view of the current therapies.
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Fusco, Anna F., Angela L. McCall, Justin S. Dhindsa, Lucy Zheng, Aidan Bailey, Amanda F. Kahn, and Mai K. ElMallah. "The Respiratory Phenotype of Pompe Disease Mouse Models." International Journal of Molecular Sciences 21, no. 6 (March 24, 2020): 2256. http://dx.doi.org/10.3390/ijms21062256.
Full textAbstract:
Pompe disease is a glycogen storage disease caused by a deficiency in acid α-glucosidase (GAA), a hydrolase necessary for the degradation of lysosomal glycogen. This deficiency in GAA results in muscle and neuronal glycogen accumulation, which causes respiratory insufficiency. Pompe disease mouse models provide a means of assessing respiratory pathology and are important for pre-clinical studies of novel therapies that aim to treat respiratory dysfunction and improve quality of life. This review aims to compile and summarize existing manuscripts that characterize the respiratory phenotype of Pompe mouse models. Manuscripts included in this review were selected utilizing specific search terms and exclusion criteria. Analysis of these findings demonstrate that Pompe disease mouse models have respiratory physiological defects as well as pathologies in the diaphragm, tongue, higher-order respiratory control centers, phrenic and hypoglossal motor nuclei, phrenic and hypoglossal nerves, neuromuscular junctions, and airway smooth muscle. Overall, the culmination of these pathologies contributes to severe respiratory dysfunction, underscoring the importance of characterizing the respiratory phenotype while developing effective therapies for patients.
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Zhang, Ran, Yuan T. Pan, Shouming He, Michael Lam, Gary D. Brayer, Alan D. Elbein, and Stephen G. Withers. "Mechanistic Analysis of Trehalose Synthase from Mycobacterium smegmatis." Journal of Biological Chemistry 286, no. 41 (August 12, 2011): 35601–9. http://dx.doi.org/10.1074/jbc.m111.280362.
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Trehalose synthase (TreS) catalyzes the reversible interconversion of maltose and trehalose and has been shown recently to function primarily in the mobilization of trehalose as a glycogen precursor. Consequently, the mechanism of this intriguing isomerase is of both academic and potential pharmacological interest. TreS catalyzes the hydrolytic cleavage of α-aryl glucosides as well as α-glucosyl fluoride, thereby allowing facile, continuous assays. Reaction of TreS with 5-fluoroglycosyl fluorides results in the trapping of a covalent glycosyl-enzyme intermediate consistent with TreS being a member of the retaining glycoside hydrolase family 13 enzyme family, thus likely following a two-step, double displacement mechanism. This trapped intermediate was subjected to protease digestion followed by LC-MS/MS analysis, and Asp230 was thereby identified as the catalytic nucleophile. The isomerization reaction was shown to be an intramolecular process by demonstration of the inability of TreS to incorporate isotope-labeled exogenous glucose into maltose or trehalose consistent with previous studies on other TreS enzymes. The absence of a secondary deuterium kinetic isotope effect and the general independence of kcat upon leaving group ability both point to a rate-determining conformational change, likely the opening and closing of the enzyme active site.
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Mizanur, Rahman M., Amanda K. K. Griffin, and Nicola L. Pohl. "Recombinant production and biochemical characterization of a hyperthermostable α-glucan/maltodextrin phosphorylase fromPyrococcus furiosus." Archaea 2, no. 3 (2008): 169–76. http://dx.doi.org/10.1155/2008/549759.
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Alpha-glucan phosphorylase catalyzes the reversible cleavage of α-1-4-linked glucose polymers into α-D-glucose-1-phosphate. We report the recombinant production of an α-glucan/maltodextrin phosphorylase (PF1535) from a hyperthermophilic archaeon,Pyrococcus furiosus, and the first detailed biochemical characterization of this enzyme from any archaeal source using a mass-spectrometry-based assay. The apparent 98 kDa recombinant enzyme was active over a broad range of temperatures and pH, with optimal activity at 80 °C and pH 6.5–7. This archaeal protein retained its complete activity after 24 h at 80 °C in Tris-HCl buffer. Unlike other previously reported phosphorylases, the Ni-affinity column purified enzyme showed broad substrate specificity in both the synthesis and degradation of maltooligosaccharides. In the synthetic direction of the enzymatic reaction, the lowest oligosaccharide required for the chain elongation was maltose. In the degradative direction, the archaeal enzyme can produce glucose-1-phosphate from maltotriose or longer maltooligosaccharides including both glycogen and starch. The specific activity of the enzyme at 80 °C in the presence of 10 mM maltoheptaose and at 10 mg ml–1glycogen concentration was 52 U mg–1and 31 U mg–1, respectively. The apparent Michaelis constant and maximum velocity for inorganic phosphate were 31 ± 2 mM and 0.60 ± 0.02 mM min–1µg–1, respectively. An initial velocity study of the enzymatic reaction indicated a sequential bi-bi catalytic mechanism. Unlike the more widely studied mammalian glycogen phosphorylase, thePyrococcusenzyme is active in the absence of added AMP.
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Jacob, Avraham I., Miriam Horovitz-Fried, Shlomit Aga-Mizrachi, Tamar Brutman-Barazani, Hana Okhrimenko, Yehiel Zick, Chaya Brodie, and Sanford R. Sampson. "The regulatory domain of protein kinase C delta positively regulates insulin receptor signaling." Journal of Molecular Endocrinology 44, no. 3 (December 1, 2009): 155–69. http://dx.doi.org/10.1677/jme-09-0119.
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Protein kinase C delta (PKCδ) is induced by insulin to rapidly associate with insulin receptor (IR) and upregulates insulin signaling. We utilized specific JM and CT receptor domains and chimeras of PKCα and PKCδ regulatory and catalytic domains to elucidate which components of PKCδ are responsible for positive regulatory effects of PKCδ on IR signaling. Studies were performed on L6 and L8 skeletal muscle myoblasts and myotubes. PKCδ was preferentially bound to the JM domain of IR, and insulin stimulation increased this binding. Both PKCδ/α and PKCα/δ chimeras (regulatory/catalytic) were bound preferentially to the JM but not to the CT domain of IR. Although IR–PKCδ binding was higher in cells expressing either the PKCδ/α or PKCα/δ chimera than in control cells, upregulation of IR signaling was observed only in PKCδ/α cells. Thus, in response to insulin increases in tyrosine phosphorylation of IR and insulin receptor substrate-1, downstream signaling to protein kinase B and glycogen synthase kinase 3 (GSK3) and glucose uptake were greater in cells overexpressing PKCδ/α and the PKCδ/δ domains than in cells expressing the PKCα/δ domains. Basal binding of Src to PKCδ was higher in both PKCδ/α- and PKCα/δ-expressing cells compared to control. Binding of Src to IR was decreased in PKCα/δ cells but remained elevated in the PKCδ/α cells in response to insulin. Finally, insulin increased Src activity in PKCδ/α-expressing cells but decreased it in PKCα/δ-expressing cells. Thus, the regulatory domain of PKCδ via interaction with Src appears to determine the role of PKCδ as a positive regulator of IR signaling in skeletal muscle.
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Sitarz, Katarzyna, Krzysztof Czamara, Joanna Bialecka, Malgorzata Klimek, Barbara Zawilinska, Slawa Szostek, and Agnieszka Kaczor. "HPV Infection Significantly Accelerates Glycogen Metabolism in Cervical Cells with Large Nuclei: Raman Microscopic Study with Subcellular Resolution." International Journal of Molecular Sciences 21, no. 8 (April 11, 2020): 2667. http://dx.doi.org/10.3390/ijms21082667.
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Using Raman microscopy, we investigated epithelial cervical cells collected from 96 women with squamous cell carcinoma (SCC) or belonging to groups I, IIa, IIID-1 and IIID-2 according to Munich III classification (IIID-1 and IIID-2 corresponding to Bethesda LSIL and HSIL groups, respectively). All women were tested for human papillomavirus (HPV) infection using PCR. Subcellular resolution of Raman microscopy enabled to understand phenotypic differences in a heterogeneous population of cervical cells in the following groups: I/HPV−, IIa/HPV−, IIa/HPV−, LSIL/HPV−, LSIL/HPV+, HSIL/HPV−, HSIL/HPV+ and cancer cells (SCC/HPV+). We showed for the first time that the glycogen content in the cytoplasm decreased with the nucleus size of cervical cells in all studied groups apart from the cancer group. For the subpopulation of large-nucleus cells HPV infection resulted in considerable glycogen depletion compared to HPV negative cells in IIa, LSIL (for both statistical significance, ca. 45%) and HSIL (trend, 37%) groups. We hypothesize that accelerated glycogenolysis in large-nucleus cells may be associated with the increased protein metabolism for HPV positive cells. Our work underlines unique capabilities of Raman microscopy in single cell studies and demonstrate potential of Raman-based methods in HPV diagnostics.
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Oetjen, E., C. Schweickhardt, K. Unthan-Fechner, and I. Probst. "Stimulation of glucose production from glycogen by glucagon, noradrenaline and non-degradable adenosine analogues is counteracted by adenosine and ATP in cultured rat hepatocytes." Biochemical Journal 271, no. 2 (October 15, 1990): 337–44. http://dx.doi.org/10.1042/bj2710337.
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The glycogenolytic potency of adenosine and ATP was studied in adult rat hepatocytes and compared with the action of glucagon and noradrenaline. In cells cultured for 48 h, adenosine and ATP as well as their analogues 2-chloroadenosine, phenylisopropyladenosine, N-ethylcarboxamidoadenosine and beta-gamma-methylene-substituted ATP (p[CH2]ppA) increased glycogen phosphorylase alpha to levels indistinguishable from those obtained by the addition of glucagon or noradrenaline. The P1 receptor antagonist 8-phenyltheophylline abolished the activation of phosphorylase by adenosine and by p[CH2]ppA, but not that by ATP. Protein kinase A was activated by p[CH2]ppA and ATP via their breakdown to adenosine. [14C]Glucose production from glycogen was stimulated only 3-fold by ATP and adenosine, compared with a 7-fold increase produced by the hormones. Stimulation of glucose production by glucagon or noradrenaline was almost completely abolished by ATP or adenosine, with half-maximal effects at around 10 microM. The non-degradable adenosine analogues were equipotent with glucagon with respect to stimulation of glucose production, and their action was also inhibited by adenosine. ATP and p[CH2]ppA, which were both degraded to adenosine, showed comparable metabolic effects, whereas the alpha, beta-methylene analogue was without biological action and also was not degraded to adenosine. In the presence of the adenosine transport inhibitor nitrobenzyl thioinosine (NBTI), adenosine exerted an increased glycogenolytic potency, reaching 80% of the maximal stimulation obtained by glucagon. The glucagon-antagonistic effect of adenosine could be completely abolished by NBTI, but was not affected by phenyltheophylline. It is concluded that, in the hepatocyte culture system, adenosine and ATP decrease the catalytic efficiency of phosphorylase alpha through signals arising from their uptake into the cell.
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Bowdish, K. S., H. E. Yuan, and A. P. Mitchell. "Analysis of RIM11, a yeast protein kinase that phosphorylates the meiotic activator IME1." Molecular and Cellular Biology 14, no. 12 (December 1994): 7909–19. http://dx.doi.org/10.1128/mcb.14.12.7909.
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Many yeast genes that are essential for meiosis are expressed only in meiotic cells. Known regulators of early meiotic genes include IME1, which is required for their expression, and SIN3 and UME6, which prevent their expression in nonmeiotic cells. We report here the molecular characterization of the RIM11 gene, which we find is required for expression of several early meiotic genes. A close functional relationship between RIM11 and IME1 is supported by two observations. First, sin3 and ume6 mutations are epistatic to rim11 mutations; prior studies have demonstrated their epistasis to ime1 mutations. Second, overexpression of RIM11 can suppress an ime1 missense mutation (ime1-L321F) but not an ime1 deletion. Sequence analysis indicates that RIM11 specifies a protein kinase related to rat glycogen synthase kinase 3 and the Drosophila shaggy/zw3 gene product. Three partially defective rim11 mutations alter residues involved in ATP binding or catalysis, and a completely defective rim11 mutation alters a tyrosine residue that corresponds to the site of an essential phosphorylation for glycogen synthase kinase 3. Immune complexes containing a hemagglutinin (HA) epitope-tagged RIM11 derivative, HA-RIM11, phosphorylate two proteins, p58 and p60, whose biological function is undetermined. In addition, HA-RIM11 immune complexes phosphorylate a functional IME1 derivative but not the corresponding ime1-L321F derivative. We propose that RIM11 stimulates meiotic gene expression through phosphorylation of IME1.
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Bowdish, K. S., H. E. Yuan, and A. P. Mitchell. "Analysis of RIM11, a yeast protein kinase that phosphorylates the meiotic activator IME1." Molecular and Cellular Biology 14, no. 12 (December 1994): 7909–19. http://dx.doi.org/10.1128/mcb.14.12.7909-7919.1994.
Full textAbstract:
Many yeast genes that are essential for meiosis are expressed only in meiotic cells. Known regulators of early meiotic genes include IME1, which is required for their expression, and SIN3 and UME6, which prevent their expression in nonmeiotic cells. We report here the molecular characterization of the RIM11 gene, which we find is required for expression of several early meiotic genes. A close functional relationship between RIM11 and IME1 is supported by two observations. First, sin3 and ume6 mutations are epistatic to rim11 mutations; prior studies have demonstrated their epistasis to ime1 mutations. Second, overexpression of RIM11 can suppress an ime1 missense mutation (ime1-L321F) but not an ime1 deletion. Sequence analysis indicates that RIM11 specifies a protein kinase related to rat glycogen synthase kinase 3 and the Drosophila shaggy/zw3 gene product. Three partially defective rim11 mutations alter residues involved in ATP binding or catalysis, and a completely defective rim11 mutation alters a tyrosine residue that corresponds to the site of an essential phosphorylation for glycogen synthase kinase 3. Immune complexes containing a hemagglutinin (HA) epitope-tagged RIM11 derivative, HA-RIM11, phosphorylate two proteins, p58 and p60, whose biological function is undetermined. In addition, HA-RIM11 immune complexes phosphorylate a functional IME1 derivative but not the corresponding ime1-L321F derivative. We propose that RIM11 stimulates meiotic gene expression through phosphorylation of IME1.
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Delqué Bayer, P., C. Vittori, P. Sudaka, and J. Giudicelli. "Purification and properties of neutral maltase from human granulocytes." Biochemical Journal 263, no. 3 (November 1, 1989): 647–52. http://dx.doi.org/10.1042/bj2630647.
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A procedure for the purification of neutral maltase from human polymorphonuclear leukocytes is described, involving solubilization with Triton X-100, proteolytic attack and three chromatographic steps: DEAE ion exchange, AcA 22 gel filtration and a second DEAE chromatography. The enzyme was obtained with a final specific activity of 30 units/mg of protein, comparable with that of other neutral maltases previously purified. The Mr of the enzyme was 550,000 as determined by gel filtration. SDS/polyacrylamide-gel electrophoresis, under non-denaturing conditions, led to a major band of 500,000 and a minor one of 260,000, both active, suggesting a polymeric or aggregated form of the protein. The catalytic properties of the human granulocytic neutral maltase were investigated. The pH optimum was around 6. The enzyme exhibited a broad range of substrate specificity, hydrolysing di- and oligosaccharides with alpha (1→2), alpha (1→3) and alpha (1→4) glucosidic linkages. The highest activities were observed for alpha (1→4) glucose oligomers of three to five residues. It was also found to hydrolyse polysaccharides such as starch and glycogen. The results of the inhibition studies are interpreted in terms of the existence of a large site including several subsites. The enzyme properties are broadly similar to those observed for other purified neutral alpha-glucosidases, in particular that of human kidney origin.
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Bilancio, Antonio, Klaus Okkenhaug, Montserrat Camps, Juliet L. Emery, Thomas Ruckle, Christian Rommel, and Bart Vanhaesebroeck. "Key role of the p110δ isoform of PI3K in B-cell antigen and IL-4 receptor signaling: comparative analysis of genetic and pharmacologic interference with p110δ function in B cells." Blood 107, no. 2 (January 15, 2006): 642–50. http://dx.doi.org/10.1182/blood-2005-07-3041.
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AbstractMouse gene–targeting studies have documented a central role of the p110δ isoform of phosphoinositide 3-kinase (PI3K) in B-cell development and function. A defect in B-cell antigen receptor (BCR) signaling is key to this B-cell phenotype. Here we further characterize this signaling defect and report that a p110δ-selective small molecule inhibitor mirrors the effect of genetic inactivation of p110δ in BCR signaling. p110δ activity is indispensable for BCR-induced DNA synthesis and phosphorylation of Akt/protein kinase B (PKB), forkhead transcription factor/forkhead box O3a (FOXO3a), and p70 S6 kinase (p70 S6K), with modest effects on the phosphorylation of glycogen synthase kinase 3 α/β (GSK3α/β) and extracellular signal-regulated kinase (Erk). The PI3K-dependent component of intracellular calcium mobilization also completely relies on p110δ catalytic activity. Resting B cells with inactive p110δ fail to enter the cell cycle, correlating with an incapacity to up-regulate the expression of cyclins D2, A, and E, and to phosphorylate the retinoblastoma protein (Rb). p110δ is also critical for interleukin 4 (IL-4)–induced phosphorylation of Akt/PKB and FOXO3a, and protection from apoptosis. Taken together, these data show that defects observed in p110δ mutant mice are not merely a consequence of altered B-cell differentiation, and emphasize the potential utility of p110δ as a drug target in autoimmune diseases in which B cells play a crucial role.
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Mohapatra, Samar Ballabha, and Narayanan Manoj. "Structural basis of catalysis and substrate recognition by the NAD(H)-dependent α-d-glucuronidase from the glycoside hydrolase family 4." Biochemical Journal 478, no. 4 (February 26, 2021): 943–59. http://dx.doi.org/10.1042/bcj20200824.
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Members of the glycoside hydrolase family 4 (GH4) employ an unusual glycosidic bond cleavage mechanism utilizing NAD(H) and a divalent metal ion, under reducing conditions. These enzymes act upon a diverse range of glycosides, and unlike most other GH families, homologs here are known to accommodate both α- and β-anomeric specificities within the same active site. Here, we report the catalytic properties and the crystal structures of TmAgu4B, an α-d-glucuronidase from the hyperthermophile Thermotoga maritima. The structures in three different states include the apo form, the NADH bound holo form, and the ternary complex with NADH and the reaction product d-glucuronic acid, at 2.15, 1.97 and 1.85 Å resolutions, respectively. These structures reveal the step-wise route of conformational changes required in the active site to achieve the catalytically competent state, and illustrate the direct role of residues that determine the reaction mechanism. Furthermore, a structural transition of a helical region in the active site to a turn geometry resulting in the rearrangement of a unique arginine residue governs the exclusive glucopyranosiduronic acid recognition in TmAgu4B. Mutational studies show that modifications of the glycone binding site geometry lead to catalytic failure and indicate overlapping roles of specific residues in catalysis and substrate recognition. The data highlight hitherto unreported molecular features and associated active site dynamics that determine the structure–function relationships within the unique GH4 family.
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Gavete, M. L., M. A. Martín, C. Alvarez, and F. Escrivá. "Maternal Food Restriction Enhances Insulin-Induced GLUT-4 Translocation and Insulin Signaling Pathway in Skeletal Muscle from Suckling Rats." Endocrinology 146, no. 8 (August 1, 2005): 3368–78. http://dx.doi.org/10.1210/en.2004-1658.
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Abstract Restriction of protein calories during stages of immaturity has a major influence on glucose metabolism and increases the risk of type 2 diabetes in adulthood. However, it is known that reduction of food intake alleviates insulin resistance. We previously demonstrated an improved insulin-induced glucose uptake in skeletal muscle of chronically undernourished adult rats. The purpose of this work was to investigate whether this condition is present during suckling, a period characterized by physiological insulin resistance as well as elucidate some of the underlying mechanisms. With this aim, 10-d-old pups from food-restricted dams were studied. We showed that undernourished suckling rats are glucose normotolerants, despite their depressed insulin secretion capacity. The content of the main glucose transporters in muscle, GLUT-4 and GLUT-1, was not affected by undernutrition, but fractionation studies showed an improved insulin-stimulated GLUT-4 translocation. p38MAPK protein, implicated in up-regulation of intrinsic activity of translocated GLUT-4, was increased. These changes suggest an improved insulin-induced glucose uptake associated with undernutrition. Insulin receptor content as well as that of both regulatory and catalytic phosphoinositol 3-kinase subunits was increased by food restriction. Insulin receptor substrate-1-associated phosphoinositol 3-kinase activity after insulin was enhanced in undernourished rats, as was phospho-glycogen synthase kinase-3, in line with insulin hypersensitivity. Surprisingly, protein tyrosine phosphatase-1B association with insulin receptor was also increased by undernutrition. These adaptations to a condition of severely limited nutritional resources might result in changes in the development of key tissues and be detrimental later in life, when a correct amount of nutrients is available, as the thrifty phenotype hypothesis predicts.
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BIONDI, Ricardo M., and Angel R. NEBREDA. "Signalling specificity of Ser/Thr protein kinases through docking-site-mediated interactions." Biochemical Journal 372, no. 1 (May 15, 2003): 1–13. http://dx.doi.org/10.1042/bj20021641.
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Signal transduction pathways use protein kinases for the modification of protein function by phosphorylation. A major question in the field is how protein kinases achieve the specificity required to regulate multiple cellular functions. Here we review recent studies that illuminate the mechanisms used by three families of Ser/Thr protein kinases to achieve substrate specificity. These kinases rely on direct docking interactions with substrates, using sites distinct from the phospho-acceptor sequences. Docking interactions also contribute to the specificity and regulation of protein kinase activities. Mitogen-activated protein kinase (MAPK) family members can associate with and phosphorylate specific substrates by virtue of minor variations in their docking sequences. Interestingly, the same MAPK docking pocket that binds substrates also binds docking sequences of positive and negative MAPK regulators. In the case of glycogen synthase kinase 3 (GSK3), the presence of a phosphate-binding site allows docking of previously phosphorylated (primed) substrates; this docking site is also required for the mechanism of GSK3 inhibition by phosphorylation. In contrast, non-primed substrates interact with a different region of GSK3. Phosphoinositide-dependent protein kinase-1 (PDK1) contains a hydrophobic pocket that interacts with a hydrophobic motif present in all known substrates, enabling their efficient phosphorylation. Binding of the substrate hydrophobic motifs to the pocket in the kinase domain activates PDK1 and other members of the AGC family of protein kinases. Finally, the analysis of protein kinase structures indicates that the sites used for docking substrates can also bind N- and C-terminal extensions to the kinase catalytic core and participate in the regulation of its activity.
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Chou, Ching-Hsuan, and Chia-Ron Yang. "Neuroprotective Studies of Evodiamine in an Okadaic Acid-Induced Neurotoxicity." International Journal of Molecular Sciences 22, no. 10 (May 19, 2021): 5347. http://dx.doi.org/10.3390/ijms22105347.
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Background: Alzheimer’s disease (AD) is the most common neurodegenerative disease, and it manifests as progressive memory loss and cognitive decline. However, there are no effective therapies for AD, which is an urgent problem to solve. Evodiamine, one of the main bioactive ingredients of Evodia rutaecarpa, has been reported to ameliorate blood–brain barrier (BBB) permeability and improve cognitive impairment in ischemia and AD mouse models. However, whether evodiamine alleviates tauopathy remains unclear. This study aimed to examine whether evodiamine ameliorates tau phosphorylation and cognitive deficits in AD models. Methods: A protein phosphatase 2A inhibitor, okadaic acid (OA), was used to induce tau phosphorylation to mimic AD-like models in neuronal cells. Protein expression and cell apoptosis were detected using Western blotting and flow cytometry, respectively. Spatial memory/cognition was assessed using water maze, passive avoidance tests, and magnetic resonance imaging assay in OA-induced mice models, and brain slices were evaluated further by immunohistochemistry. Results: The results showed that evodiamine significantly reduced the expression of phosphor-tau, and further decreased tau aggregation and neuronal cell death in response to OA treatment. This inhibition was found to be via the inhibition of glycogen synthase kinase 3β, cyclin-dependent kinase 5, and mitogen-activated protein kinase pathways. In vivo results indicated that evodiamine treatment ameliorated learning and memory impairments in mice, whereas Western blotting and immunohistochemical analysis of the mouse brain also confirmed the neuroprotective effects of evodiamine. Conclusions: Evodiamine can decrease the neurotoxicity of tau aggregation and exhibit a neuroprotective effect. Our results demonstrate that evodiamine has a therapeutic potential for AD treatment.
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Wek, R. C., J. F. Cannon, T. E. Dever, and A. G. Hinnebusch. "Truncated protein phosphatase GLC7 restores translational activation of GCN4 expression in yeast mutants defective for the eIF-2 alpha kinase GCN2." Molecular and Cellular Biology 12, no. 12 (December 1992): 5700–5710. http://dx.doi.org/10.1128/mcb.12.12.5700.
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GCN2 is a protein kinase in Saccharomyces cerevisiae that is required for increased expression of the transcriptional activator GCN4 in amino acid-starved cells. GCN2 stimulates GCN4 synthesis at the translational level by phosphorylating the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2). We identified a truncated form of the GLC7 gene, encoding the catalytic subunit of a type 1 protein phosphatase, by its ability to restore derepression of GCN4 expression in a strain containing the partially defective gcn2-507 allele. Genetic analysis suggests that the truncated GLC7 allele has a dominant negative phenotype, reducing the level of native type 1 protein phosphatase activity in the cell. The truncated form of GLC7 does not suppress the regulatory defect associated with a gcn2 deletion or a mutation in the phosphorylation site of eIF-2 alpha (Ser-51). In addition, the presence of multiple copies of wild-type GLC7 impairs the derepression of GCN4 that occurs in response to amino acid starvation or dominant-activating mutations in GCN2. These findings suggest that the phosphatase activity of GLC7 acts in opposition to the kinase activity of GCN2 in modulating the level of eIF-2 alpha phosphorylation and the translational efficiency of GCN4 mRNA. This conclusion is supported by biochemical studies showing that the truncated GLC7 allele increases the level of eIF-2 alpha phosphorylation in the gcn2-507 mutant to a level approaching that seen in wild-type cells under starvation conditions. The truncated GLC7 allele also leads to reduced glycogen accumulation, indicating that this protein phosphatase is involved in regulating diverse metabolic pathways in yeast cells.
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Wek, R. C., J. F. Cannon, T. E. Dever, and A. G. Hinnebusch. "Truncated protein phosphatase GLC7 restores translational activation of GCN4 expression in yeast mutants defective for the eIF-2 alpha kinase GCN2." Molecular and Cellular Biology 12, no. 12 (December 1992): 5700–5710. http://dx.doi.org/10.1128/mcb.12.12.5700-5710.1992.
Full textAbstract:
GCN2 is a protein kinase in Saccharomyces cerevisiae that is required for increased expression of the transcriptional activator GCN4 in amino acid-starved cells. GCN2 stimulates GCN4 synthesis at the translational level by phosphorylating the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2). We identified a truncated form of the GLC7 gene, encoding the catalytic subunit of a type 1 protein phosphatase, by its ability to restore derepression of GCN4 expression in a strain containing the partially defective gcn2-507 allele. Genetic analysis suggests that the truncated GLC7 allele has a dominant negative phenotype, reducing the level of native type 1 protein phosphatase activity in the cell. The truncated form of GLC7 does not suppress the regulatory defect associated with a gcn2 deletion or a mutation in the phosphorylation site of eIF-2 alpha (Ser-51). In addition, the presence of multiple copies of wild-type GLC7 impairs the derepression of GCN4 that occurs in response to amino acid starvation or dominant-activating mutations in GCN2. These findings suggest that the phosphatase activity of GLC7 acts in opposition to the kinase activity of GCN2 in modulating the level of eIF-2 alpha phosphorylation and the translational efficiency of GCN4 mRNA. This conclusion is supported by biochemical studies showing that the truncated GLC7 allele increases the level of eIF-2 alpha phosphorylation in the gcn2-507 mutant to a level approaching that seen in wild-type cells under starvation conditions. The truncated GLC7 allele also leads to reduced glycogen accumulation, indicating that this protein phosphatase is involved in regulating diverse metabolic pathways in yeast cells.
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Damri, Odeya, Nofar Shemesh, and Galila Agam. "Is There Justification to Treat Neurodegenerative Disorders by Repurposing Drugs? The Case of Alzheimer’s Disease, Lithium, and Autophagy." International Journal of Molecular Sciences 22, no. 1 (December 27, 2020): 189. http://dx.doi.org/10.3390/ijms22010189.
Full textAbstract:
Lithium is the prototype mood-stabilizer used for acute and long-term treatment of bipolar disorder. Cumulated translational research of lithium indicated the drug’s neuroprotective characteristics and, thereby, has raised the option of repurposing it as a drug for neurodegenerative diseases. Lithium’s neuroprotective properties rely on its modulation of homeostatic mechanisms such as inflammation, mitochondrial function, oxidative stress, autophagy, and apoptosis. This myriad of intracellular responses are, possibly, consequences of the drug’s inhibition of the enzymes inositol-monophosphatase (IMPase) and glycogen-synthase-kinase (GSK)-3. Here we review lithium’s neurobiological properties as evidenced by its neurotrophic and neuroprotective properties, as well as translational studies in cells in culture, in animal models of Alzheimer’s disease (AD) and in patients, discussing the rationale for the drug’s use in the treatment of AD.
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Leconte, I., and E. Clauser. "Two sequences flanking the major autophosphorylation site of the insulin receptor are essential for tyrosine kinase activation." Biochemical Journal 306, no. 2 (March 1, 1995): 465–72. http://dx.doi.org/10.1042/bj3060465.
Full textAbstract:
The tyrosine kinase domain of the human insulin receptor (IR) contains several short amino acid motifs which are strictly conserved in all protein kinases and two sequence motifs which are specific to the tyrosine kinases (AAR or RAA and P(I)/VK/RWT/M). In the serine/threonine kinases these motifs are replaced by the sequences KPE and GT/SXXY/PX respectively. In the present work, the tyrosine kinase-specific sequences of the IR (1134AAR1136 and 1172PVRWM1176) were replaced using site-directed mutagenesis by sequences which confer a serine kinase specificity on the receptor. Five different IR mutants were expressed in Chinese hamster ovary (CHO) or COS cells and their structural and functional properties compared with those of the wild-type recombinant human IR. These mutants are processed normally and bind insulin with normal affinities. None of the mutants containing a putative serine kinase-specific sequence display detectable autophosphorylation or tyrosine kinase activity in response to insulin, either in vitro or in vivo. These mutants were also unable to phosphorylate serine/threonine kinase substrates after insulin stimulation. Unexpectedly, they showed impaired ATP binding, as studied by an original technique consisting of cross-linking adenosine 5′-([35S]thio)triphosphate to partially purified receptors. Finally, none of the studied mutants transmit the insulin signal necessary to stimulate either DNA or glycogen synthesis. These data provide evidence for the importance of these conserved sequences in the kinase domain for both receptor activation and kinase activity. Furthermore, they demonstrate that the exchange of sequences specific to the catalytic domain of tyrosine kinases for those specific to the serine/threonine kinases is not sufficient to confer serine/threonine specificity on the insulin receptor.
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Johnson, L. N., S.-H. Hu, and D. Barford. "Catalytic mechanism of glycogen phosphorylase." Faraday Discussions 93 (1992): 131. http://dx.doi.org/10.1039/fd9929300131.
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Jozefczuk, Ewelina, Piotr Szczepaniak, Tomasz Jan Guzik, and Mateusz Siedlinski. "Silencing of Sphingosine kinase 1 Affects Maturation Pathways in Mouse Neonatal Cardiomyocytes." International Journal of Molecular Sciences 22, no. 7 (March 31, 2021): 3616. http://dx.doi.org/10.3390/ijms22073616.
Full textAbstract:
Sphingosine kinase-1 (Sphk1) and its product, sphingosine-1-phosphate (S1P) are important regulators of cardiac growth and function. Numerous studies have reported that Sphk1/S1P signaling is essential for embryonic cardiac development and promotes pathological cardiac hypertrophy in adulthood. However, no studies have addressed the role of Sphk1 in postnatal cardiomyocyte (CM) development so far. The present study aimed to assess the molecular mechanism(s) by which Sphk1 silencing might influence CMs development and hypertrophy in vitro. Neonatal mouse CMs were transfected with siRNA against Sphk1 or negative control, and subsequently treated with 1 µM angiotensin II (AngII) or a control buffer for 24 h. The results of RNASeq analysis revealed that diminished expression of Sphk1 significantly accelerated neonatal CM maturation by inhibiting cell proliferation and inducing developmental pathways in the stress (AngII-induced) conditions. Importantly, similar effects were observed in the control conditions. Enhanced maturation of Sphk1-lacking CMs was further confirmed by the upregulation of the physiological hypertrophy-related signaling pathway involving Akt and downstream glycogen synthase kinase 3 beta (Gsk3β) downregulation. In summary, we demonstrated that the Sphk1 silencing in neonatal mouse CMs facilitated their postnatal maturation in both physiological and stress conditions.
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Abd El-Fadeal, Noha M., Mohamed S. Nafie, Mohammed K. El-kherbetawy, Amr El-mistekawy, Hala M. F. Mohammad, Alaaeldeen M. Elbahaie, Abdullah A. Hashish, et al. "Antitumor Activity of Nitazoxanide against Colon Cancers: Molecular Docking and Experimental Studies Based on Wnt/β-Catenin Signaling Inhibition." International Journal of Molecular Sciences 22, no. 10 (May 14, 2021): 5213. http://dx.doi.org/10.3390/ijms22105213.
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In colon cancer, wingless (Wnt)/β-catenin signaling is frequently upregulated; however, the creation of a molecular therapeutic agent targeting this pathway is still under investigation. This research aimed to study how nitazoxanide can affect Wnt/β-catenin signaling in colon cancer cells (HCT-116) and a mouse colon cancer model. Our study included 2 experiments; the first was to test the cytotoxic activity of nitazoxanide in an in vitro study on a colon cancer cell line (HCT-116) versus normal colon cells (FHC) and to highlight the proapoptotic effect by MTT assay, flow cytometry and real-time polymerase chain reaction (RT-PCR). The second experiment tested the in vivo cytotoxic effect of nitazoxanide against 1,2-dimethylhydrazine (DMH) prompted cancer in mice. Mice were grouped as saline, DMH control and DMH + nitazoxanide [100 or 200 mg per kg]. Colon levels of Wnt and β-catenin proteins were assessed by Western blotting while proliferation was measured via immunostaining for proliferating cell nuclear antigen (PCNA). Treating HCT-116 cells with nitazoxanide (inhibitory concentration 50 (IC50) = 11.07 µM) revealed that it has a more cytotoxic effect when compared to 5-flurouracil (IC50 = 11.36 µM). Moreover, it showed relatively high IC50 value (non-cytotoxic) against the normal colon cells. Nitazoxanide induced apoptosis by 15.86-fold compared to control and arrested the cell cycle. Furthermore, nitazoxanide upregulated proapoptotic proteins (P53 and BAX) and caspases but downregulated BCL-2. Nitazoxanide downregulated Wnt/β-catenin/glycogen synthase kinase-3β (GSK-3β) signaling and PCNA staining in the current mouse model. Hence, our findings highlighted the cytotoxic effect of nitazoxanide and pointed out the effect on Wnt/β-catenin/GSK-3β signaling.
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Severi, Ilenia, Marco Fosca, Georgia Colleluori, Federico Marini, Luca Imperatori, Martina Senzacqua, Angelica Di Vincenzo, et al. "High-Fat Diet Impairs Mouse Median Eminence: A Study by Transmission and Scanning Electron Microscopy Coupled with Raman Spectroscopy." International Journal of Molecular Sciences 22, no. 15 (July 28, 2021): 8049. http://dx.doi.org/10.3390/ijms22158049.
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Hypothalamic dysfunction is an initial event following diet-induced obesity, primarily involving areas regulating energy balance such as arcuate nucleus (Arc) and median eminence (ME). To gain insights into the early hypothalamic diet-induced alterations, adult CD1 mice fed a high-fat diet (HFD) for 6 weeks were studied and compared with normo-fed controls. Transmission and scanning electron microscopy and histological staining were employed for morphological studies of the ME, while Raman spectroscopy was applied for the biochemical analysis of the Arc-ME complex. In HFD mice, ME β2-tanycytes, glial cells dedicated to blood-liquor crosstalk, exhibited remarkable ultrastructural anomalies, including altered alignment, reduced junctions, degenerating organelles, and higher content of lipid droplets, lysosomes, and autophagosomes. Degenerating tanycytes also displayed an electron transparent cytoplasm filled with numerous vesicles, and they were surrounded by dilated extracellular spaces extending up to the subependymal layer. Consistently, Raman spectroscopy analysis of the Arc-ME complex revealed higher glycogen, collagen, and lipid bands in HFD mice compared with controls, and there was also a higher band corresponding to the cyanide group in the former compared to the last. Collectively, these data show that ME β2-tanycytes exhibit early structural and chemical alterations due to HFD and reveal for the first-time hypothalamic cyanide presence following high dietary lipids consumption, which is a novel aspect with potential implications in the field of obesity.
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Hajikarimlou, Maryam, Kathryn Hunt, Grace Kirby, Sarah Takallou, Sasi Kumar Jagadeesan, Katayoun Omidi, Mohsen Hooshyar, et al. "Lithium Chloride Sensitivity in Yeast and Regulation of Translation." International Journal of Molecular Sciences 21, no. 16 (August 10, 2020): 5730. http://dx.doi.org/10.3390/ijms21165730.
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For decades, lithium chloride (LiCl) has been used as a treatment option for those living with bipolar disorder (BD). As a result, many studies have been conducted to examine its mode of action, toxicity, and downstream cellular responses. We know that LiCl is able to affect cell signaling and signaling transduction pathways through protein kinase C and glycogen synthase kinase-3, which are considered to be important in regulating gene expression at the translational level. However, additional downstream effects require further investigation, especially in translation pathway. In yeast, LiCl treatment affects the expression, and thus the activity, of PGM2, a phosphoglucomutase involved in sugar metabolism. Inhibition of PGM2 leads to the accumulation of intermediate metabolites of galactose metabolism causing cell toxicity. However, it is not fully understood how LiCl affects gene expression in this matter. In this study, we identified three genes, NAM7, PUS2, and RPL27B, which increase yeast LiCl sensitivity when deleted. We further demonstrate that NAM7, PUS2, and RPL27B influence translation and exert their activity through the 5′-Untranslated region (5′-UTR) of PGM2 mRNA in yeast.
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Benajiba, Lina, Florence Wagner, Linda Ross, Ilene Galinsky, Daniel J. DeAngelo, Jennifer Gale, Jen Pan, et al. "Identification of a First in Class GSK3-Alpha Selective Inhibitor As a New Differentiation Therapy for AML." Blood 126, no. 23 (December 3, 2015): 870. http://dx.doi.org/10.1182/blood.v126.23.870.870.
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Abstract Isoform-selective enzyme targeting poses a potential clinical challenge when there is significant homology in the protein activity domains. Glycogen synthase kinase 3-alpha (GSK3α) has been identified previously as a therapeutic target to promote differentiation in acute myeloid leukemia (AML) (Banerji et al, JCI, 2012). The GSK3 family consists of two highly homologous, but functionally non-redundant, isoforms (α and β). These proteins are serine-threonine kinases implicated in a diversity of cellular processes. Concurrent inhibition of both isoforms is known to induce β-catenin stabilization and can therefore increase self-renewal of leukemia initiating cells. Isoform-selective GSK3 suppression does not increase β-catenin levels (Doble et al, Dev Cell, 2007) and thus may offer a new therapeutic approach. GSK3α selective inhibitors, with cell-based activity, have not been reported. Our collaborative study aimed to identify first-in-class GSK3α selective inhibitors and evaluate their activity in AML, a disease in great need for new therapeutic approaches. A series of potent and selective inhibitors for GSK3α/β were discovered through a screening campaign supported by the NIH's Molecular Libraries Probe Centers Network (MLPCN) program. While these compounds are equipotent for GSK3α/β, they demonstrate remarkable selectivity versus the larger kinome (> 100x selective vs 311 kinases). Analysis of the high resolution x-ray crystal structure obtained with BRD9421, one of the lead compounds, and GSK3β revealed a tridentate hydrogen bond binding mode within the ATP catalytic domain. One of these interactions, is to aspartic 133 in GSK3β. This same residue in GSK3α is a glutamic acid and represents one of the two amino acid differences within the ATP binding domain between these two enzymes. GSK3α and β share 98% homology in their catalytic domains, but the exploitation of this hydrogen bond interaction through rational drug design offered an excellent opportunity to produce selective inhibitors for GSK3α and GSK3β. BRD0705 was thus identified as an 8-fold GSK3α isoform-selective inhibitor over GSK3β (IC50 GSK3α: 0.045 vs. GSK3β: 0.350 μM). We first validated BRD0705 isoform-selectivity in a panel of AML cell lines and primary patients (n=3 cell lines and n= 2 patient samples). Treatment with BRD0705 resulted in a dose- and time-dependent decrease in tyr279 GSK3α phosphorylation without any effect on tyr216 GSK3β phosphorylation. No increase in β-catenin protein levels by western blotting and no nuclear localization by immunofluorescence were observed. GSK3α inhibition with BRD0705 led to morphological and surface marker changes (CD11b, CD11c, CD14, CD117) consistent with AML differentiation in a panel of AML cell lines (n=4) and primary AML samples (n=5). BRD0705 also induced a G2/M arrest and impaired methylcellulose colony formation in AML cell lines and primary patient samples. BRD0705 possesses favorable pharmacokinetic properties and was well tolerated without any observed adverse events in C57BL/6 mice with repeated dosing. In vivo efficacy studies in AML models are ongoing. In conclusion, we identified a first-in-class GSK3α isoform-selective inhibitor and conducted preclinical studies validating BRD0705 as a promising new differentiation therapeutic candidate in AML. Small-molecule inhibition of GSK3α induced myeloid differentiation, cell cycle arrest and impaired colony formation without β-catenin stabilization in AML. Disclosures DeAngelo: Incyte: Consultancy; Novartis: Consultancy; Amgen: Consultancy; Pfizer: Consultancy; Ariad: Consultancy; Agios: Consultancy; Bristol Myers Squibb: Consultancy; Celgene: Consultancy. Stone:Sunesis: Consultancy, Other: DSMB for clinical trial; Roche/Genetech: Consultancy; Agios: Consultancy; Karyopharm: Consultancy; Merck: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Novartis: Research Funding; Celator: Consultancy; Amgen: Consultancy; Pfizer: Consultancy; AROG: Consultancy; Juno: Consultancy. Stegmaier:Novartis Pharmaceuticals: Consultancy.