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Experimental and in silico analyses of glycolytic flux control in bloodstream form Trypanosoma brucei.

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Albert, Marie-Astrid [UCL] Haanstra, Jurgen R Hannaert, Véronique [UCL] Van Roy, Joris [UCL] Opperdoes, Frederik [UCL] Michels, Paulus [UCL]

A mathematical model of glycolysis in bloodstream form Trypanosoma brucei was developed previously on the basis of all available enzyme kinetic data (Bakker, B. M., Michels, P. A. M., Opperdoes, F. R., and Westerhoff, H. V. (1997) J. Biol. Chem. 272, 3207-3215). The model predicted correctly the fluxes and cellular metabolite concentrations as measured in non-growing trypanosomes and the major contribution to the flux control exerted by the plasma membrane glucose transporter. Surprisingly, a large overcapacity was predicted for hexokinase (HXK), phosphofructokinase (PFK), and pyruvate kinase (PYK). Here, we present our further analysis of the control of glycolytic flux in bloodstream form T. brucei. First, the model was optimized and extended with recent information about the kinetics of enzymes and their activities as measured in lysates of in vitro cultured growing trypanosomes. Second, the concentrations of five glycolytic enzymes (HXK, PFK, phosphoglycerate mutase, enolase, and PYK) in trypanosomes were changed by RNA interference. The effects of the knockdown of these enzymes on the growth, activities, and levels of various enzymes and glycolytic flux were studied and compared with model predictions. Data thus obtained support the conclusion from the in silico analysis that HXK, PFK, and PYK are in excess, albeit less than predicted. Interestingly, depletion of PFK and enolase had an effect on the activity (but not, or to a lesser extent, expression) of some other glycolytic enzymes. Enzymes located both in the glycosomes (the peroxisome-like organelles harboring the first seven enzymes of the glycolytic pathway of trypanosomes) and in the cytosol were affected. These data suggest the existence of novel regulatory mechanisms operating in trypanosome glycolysis.

Document type Article de périodique (Journal article) – Article de recherche
Access type Accès restreint
Publication date 2005
Journal information "Journal of Biological Chemistry" - Vol. 280, no. 31, p. 28306-28315 (2005)
Peer reviewed yes
Publisher American Society for Biochemistry and Molecular Biology, Inc.
issn 0021-9258
e-issn 1083-351X
Publication status Publié
Affiliation UCL - MD/BICL - Département de biochimie et de biologie cellulaire
MESH Subject Animals ; Antiporters - genetics ; Bacterial Proteins - genetics ; Blood - parasitology ; Cell Line ; Escherichia coli - genetics ; Genes, Reporter ; Glycolysis ; Kinetics ; Models, Biological ; Polymerase Chain Reaction ; RNA, Messenger - genetics ; RNA, Protozoan - genetics ; RNA, Small Interfering ; Transfection ; Trypanosoma brucei brucei - isolation & purification - metabolism
Links
  1. Barrett Michael P, Burchmore Richard JS, Stich August, Lazzari Julio O, Frasch Alberto Carlos, Cazzulo Juan José, Krishna Sanjeev, The trypanosomiases, 10.1016/s0140-6736(03)14694-6
  2. Bakker Barbara M., Michels Paul A. M., Opperdoes Fred R., Westerhoff Hans V., Glycolysis in Bloodstream FormTrypanosoma bruceiCan Be Understood in Terms of the Kinetics of the Glycolytic Enzymes, 10.1074/jbc.272.6.3207
  3. Symp. Soc. Exp. Biol., 27, 65 (1973)
  4. Heinrich Reinhart, Rapoport Tom A., A Linear Steady-State Treatment of Enzymatic Chains. General Properties, Control and Effector Strength, 10.1111/j.1432-1033.1974.tb03318.x
  5. Fell, D. (1997) Frontiers in Metabolism: Understanding the Control of Metabolism, Vol. 2, Portland Press Ltd., London
  6. Bakker Barbara M., Michels Paul A. M., Opperdoes Fred R., Westerhoff Hans V., What Controls Glycolysis in Bloodstream FormTrypanosoma brucei?, 10.1074/jbc.274.21.14551
  7. HELFERT Sandra, ESTÉVEZ Antonio M., BAKKER Barbara, MICHELS Paul, CLAYTON Christine, Roles of triosephosphate isomerase and aerobic metabolism in Trypanosoma brucei, 10.1042/0264-6021:3570117
  8. Bakker Barbara M, Westerhoff Hans V, Opperdoes Fred R, Michels Paul A.M, Metabolic control analysis of glycolysis in trypanosomes as an approach to improve selectivity and effectiveness of drugs, 10.1016/s0166-6851(99)00197-8
  9. Boiteux A., Hess B., Design of Glycolysis, 10.1098/rstb.1981.0056
  10. Rapoport Tom A., Heinrich Reinhart, Jacobasch Gisela, Rapoport Samuel, A Linear Steady-State Treatment of Enzymatic Chains. A Mathematical Model of Glycolysis of Human Erythrocytes, 10.1111/j.1432-1033.1974.tb03320.x
  11. Stryer, L. (1995) Biochemistry, 4th Ed., W. H. Freeman & Co., New York
  12. Michels P.A.M., Hannaert V., Bringaud F., Metabolic Aspects of Glycosomes in Trypanosomatidae – New Data and Views, 10.1016/s0169-4758(00)01810-x
  13. Bakker B. M., Walsh M. C., ter Kuile B. H., Mensonides F. I. C., Michels P. A. M., Opperdoes F. R., Westerhoff H. V., Contribution of glucose transport to the control of the glycolytic flux in Trypanosoma brucei, 10.1073/pnas.96.18.10098
  14. GRUENBERG Jean, SHARMA Pukh Raj, DESHUSSES Jacques, d-Glucose Transport in Trypanosoma brucei. d-Glucose Transport is the Rate-Limiting Step of Its Metabolism, 10.1111/j.1432-1033.1978.tb12549.x
  15. Eisenthal R., Game S., Holman G.D., Specificity and kinetics of hexose transport in Trypanosoma brucei, 10.1016/0005-2736(89)90107-7
  16. J. Biol. Chem., 266, 857 (1991)
  17. SEYFANG Andreas, DUSZENKO Michael, Specificity of glucose transport in Trypanosoma brucei. Effective inhibition by phloretin and cytochalasin B, 10.1111/j.1432-1033.1991.tb16362.x
  18. Chevalier Nathalie, Rigden Daniel J., Van Roy Joris, Opperdoes Fred R., Michels Paul A. M., Trypanosoma brucei contains a 2,3-bisphosphoglycerate independent phosphoglycerate mutase : Trypanosoma brucei phosphoglycerate mutase, 10.1046/j.1432-1327.2000.01145.x
  19. Králová Ivica, Rigden Daniel J., Opperdoes Fred R., Michels Paul A. M., Glycerol kinase of Trypanosoma brucei : Cloning, molecular characterization and mutagenesis, 10.1046/j.1432-1327.2000.01238.x
  20. Hannaert Veronique, Albert Marie-Astrid, Rigden Daniel J., Theresa da Silva Giotto M., Thiemann Otavio, Garratt Richard C., Van Roy Joris, Opperdoes Fred R., Michels Paul A. M., Kinetic characterization, structure modelling studies and crystallization of Trypanosoma brucei enolase, 10.1046/j.1432-1033.2003.03692.x
  21. Guerra Daniel G., Vertommen Didier, Fothergill-Gilmore Linda A., Opperdoes Fred R., Michels Paul A. M., Characterization of the cofactor-independent phosphoglycerate mutase from Leishmania mexicana mexicana. Histidines that coordinate the two metal ions in the active site show different susceptibilities to irreversible chemical modification, 10.1111/j.1432-1033.2004.04097.x
  22. Biebinger Susanne, Elizabeth Wirtz L., Lorenz Patrick, Christine Clayton, Vectors for inducible expression of toxic gene products in bloodstream and procyclic Trypanosoma brucei, 10.1016/s0166-6851(96)02815-0
  23. Hannaert, V., Bringaud, F., Opperdoes, F. R., and Michels, P. A. M. (2003) Kinetoplastid Biology and Disease http://www.kinetoplastids.com/content/2/1/11
  24. Sambrook, J., and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  25. van den Hoff Maurice J.B., Moorman Antoon F.M., Lamers Wouter H., Electroporation in ‘intracellular’ buffer increases cell survival, 10.1093/nar/20.11.2902
  26. Methods Mol. Biol., 262, 53 (2004)
  27. MISSET Onno, OPPERDOES Fred R., Simultaneous purification of hexokinase, class-I fructose-bisphosphate aldolase, triosephosphate isomerase and phosphoglycerate kinase from Trypanosoma brucei, 10.1111/j.1432-1033.1984.tb08490.x
  28. Callens Mia, Kuntz Douglas A., Opperdoes Fred R., Characterization of pyruvate kinase of Trypanosoma brucei and its role in the regulation of carbohydrate metabolism, 10.1016/0166-6851(91)90144-u
  29. Bontemps Françoise, Hue Louis, Hers Henri-Géry, Phosphorylation of glucose in isolated rat hepatocytes. Sigmoidal kinetics explained by the activity of glucokinase alone, 10.1042/bj1740603
  30. Sauro, H. M. (2000) Proceedings of the 9th International Meeting on BioThermoKinetics: Jarnac, a System for Interactive Metabolic Analysis, in Animating the Cellular Map (Hofmeyr, J. H. S., Rohwer, J. M., and Snoep, J. L., eds) Stellenbosch University Press, Stellenbosch, South Africa
  31. Sauro Herbert M., Hucka Michael, Finney Andrew, Wellock Cameron, Bolouri Hamid, Doyle John, Kitano Hiroaki, Next Generation Simulation Tools: The Systems Biology Workbench and BioSPICE Integration, 10.1089/153623103322637670
  32. Eisenthal Robert, Panes Alison, The aerobic/anaerobic transition of glucose metabolism inTrypanosoma brucei, 10.1016/0014-5793(85)81106-6
  33. FAIRLAMB ALAN H., OPPERDOES FRED R., BORST PIET, New approach to screening drugs for activity against African trypanosomes, 10.1038/265270a0
  34. Opperdoes Fred R., Borst Piet, Localization of nine glycolytic enzymes in a microbody-like organelle inTrypanosoma brucei: The glycosome, 10.1016/0014-5793(77)80476-6
  35. Wirtz E, Clayton C, Inducible gene expression in trypanosomes mediated by a prokaryotic repressor, 10.1126/science.7761835
  36. Mol. Biochem. Parasitol., 104, 106 (1999)
  37. Chen Yili, Hung Chien-Hui, Burderer Thomas, Lee Gwo-Shu Mary, Development of RNA interference revertants in Trypanosoma brucei cell lines generated with a double stranded RNA expression construct driven by two opposing promoters, 10.1016/s0166-6851(02)00276-1
  38. Durand-Dubief Mickaël, Kohl Linda, Bastin Philippe, Efficiency and specificity of RNA interference generated by intra- and intermolecular double stranded RNA in Trypanosoma brucei, 10.1016/s0166-6851(03)00071-9
  39. Acta Trop., 39, 61 (1982)
  40. Cronin C N, Tipton K F, Purification and regulatory properties of phosphofructokinase fromTrypanosoma (Trypanozoon) brucei brucei, 10.1042/bj2270113
  41. López Claudia, Chevalier Nathalie, Hannaert Véronique, Rigden Daniel J., Michels Paul A. M., Ramirez Jose Luis, Leishmania donovani phosphofructokinase : Gene characterization, biochemical properties and structure-modelling studies, 10.1046/j.1432-1033.2002.03086.x
  42. Bakker B. M., Mensonides F. I. C., Teusink B., van Hoek P., Michels P. A. M., Westerhoff H. V., Compartmentation protects trypanosomes from the dangerous design of glycolysis, 10.1073/pnas.030539197
  43. Oduro K.K., Flynn I.W., Bowman I.B.R., Trypanosoma brucei: Activities and subcellular distribution of glycolytic enzymes from differently disrupted cells, 10.1016/0014-4894(80)90014-4
  44. Verlinde Christophe L.M.J., Hannaert Véronique, Blonski Casimir, Willson Michèle, Périé Jacques J., Fothergill-Gilmore Linda A., Opperdoes Fred R., Gelb Michael H., Hol Wim G.J., Michels Paul A.M., Glycolysis as a target for the design of new anti-trypanosome drugs, 10.1054/drup.2000.0177
  45. MISSET Onno, BOS Octaaf J. M., OPPERDOES Fred R., Glycolytic enzymes of Trypanosoma brucei. Simultaneous purification, intraglycosomal concentrations and physical properties, 10.1111/j.1432-1033.1986.tb09687.x
  46. Bakker Barbara M., Westerhoff Hans V., Michels Paul A. M., Regulation and control of compartmentalized glycolysis in bloodstream formTrypanosoma brucei, 10.1007/bf02110191
  47. Opperdoes Fred R., Michels Paul A.M., Enzymes of carbohydrate metabolism as potential drug targets, 10.1016/s0020-7519(01)00155-2
Bibliographic reference Albert, Marie-Astrid ; Haanstra, Jurgen R ; Hannaert, Véronique ; Van Roy, Joris ; Opperdoes, Frederik ; et. al. Experimental and in silico analyses of glycolytic flux control in bloodstream form Trypanosoma brucei.. In: Journal of Biological Chemistry, Vol. 280, no. 31, p. 28306-28315 (2005)
Permanent URL http://hdl.handle.net/2078.1/10220