Glycolysis in Bloodstream Form Trypanosoma brucei Can Be Understood in Terms of the Kinetics of the Glycolytic Enzymes

Bakker, Barbara M.; Michels, Paul A.M.; Opperdoes, Frederik; Westerhoff, Hans V.

doi:10.1074/jbc.272.6.3207

Cited by 193 publications

(206 citation statements)

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“…In bloodstream forms under aerobic conditions, glucose is metabolized to pyruvate and glycerol. The experimental ratio of pyruvate to glycerol ranges between 9:1 and 4:1, pending on the culture conditions used (31). To achieve this ratio, glycerol 3-phosphate (G3P) is exported from the glycosome and converted to DHAP by a mitochondrial glycerol-3-phophate dehydrogenase.…”

Section: Figmentioning

confidence: 99%

Depletion of GIM5 Causes Cellular Fragility, a Decreased Glycosome Number, and Reduced Levels of Ether-linked Phospholipids in Trypanosomes

Voncken¹,

Hellemond

Pfisterer³

et al. 2003

Journal of Biological Chemistry

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Microbody division in mammalian cells, trypanosomes, and yeast depends on the PEX11 microbody membrane proteins. The function of PEX11 is not understood, and the suggestion that it affects microbody (peroxisome) numbers in mammals and yeast, because it plays a role in beta-oxidation of fatty acids, is controversial. PEX11 and two PEX11-related proteins, GIM5A and GIM5B, are the predominant membrane proteins of the microbodies (glycosomes) of Trypanosoma brucei. The compartmentation of glycosomal enzymes is essential in trypanosomes. Deletion of the GIM5A gene from the form of the parasite that lives in the mammalian blood has no effect on trypanosome growth, but depletion of GIM5B on a gim5a null background causes death. We show here that procyclic trypanosomes, adapted for life in the Tsetse fly vector, survive without GIM5A and with very low levels of GIM5B. The depleted cells have fewer glycosomes than usual and are osmotically fragile, which is a novel observation for a microbody defect. Thus trypanosomes require both GIM5B and PEX11 for the maintenance of normal glycosome numbers. Procyclic cells lacking GIM5A, like mouse cells partially defective in PEX11, have fewer ether-linked phospholipids, even when GIM5B levels are not reduced. Metabolite measurements on GIM5A/B-depleted bloodstream form trypanosomes suggested a change in the flux through the glycolytic pathway. We conclude that PEX11 family proteins play important roles in determining microbody membrane structure, with secondary effects on a subset of microbody metabolic pathways.Microbodies are single-membrane-bound organelles found in very diverse eukaryotes; examples include the peroxisomes of mammals and yeast, the glyoxysomes and peroxisomes of plants, and the glycosomes of kinetoplastid protists. Microbodies manifest considerable diversity with regard to the enzymes and metabolic pathways they contain: examples include catalase, and enzymes of fatty acid beta-oxidation, alcohol oxidation, ether-lipid biosynthesis, and, in the kinetoplastids, glycolysis and glycerol metabolism.

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Section: Figmentioning

confidence: 99%

Depletion of GIM5 Causes Cellular Fragility, a Decreased Glycosome Number, and Reduced Levels of Ether-linked Phospholipids in Trypanosomes

Voncken¹,

Hellemond

Pfisterer³

et al. 2003

Journal of Biological Chemistry

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“…the long slender form in blood, lymphatic fluid, and cerebrospinal fluid of its mammalian host and the procyclic form in the mid-gut of the tsetse fly. Due to the lack of a functional Krebs cycle and oxidative phosphorylation capabilities, the bloodstream forms depend on glycolysis for ATP production, with glycerol as an alternative substrate (1,2). Glycolysis in trypanosomes differs markedly from the corresponding pathway in higher eukaryotes.…”

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confidence: 99%

Cloning, Heterologous Expression, and Characterization of Three Aquaglyceroporins from Trypanosoma brucei

Uzcátegui

Szallies

Pavlovic-Djuranovic³

et al. 2004

Journal of Biological Chemistry

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Trypanosoma brucei, causative for African sleeping sickness, relies exclusively on glycolysis for ATP production. Under anaerobic conditions, glucose is converted to equimolar amounts of glycerol and pyruvate, which are both secreted from the parasite. As we have shown previously, glycerol transport in T. brucei occurs via specific membrane proteins (Wille, U., Schade, B., and Duszenko, M. (1998) Eur. J. Biochem. 256, 245-250). Here, we describe cloning and biochemical characterization of the three trypanosomal aquaglyceroporins (AQP; TbAQP1-3), which show a 40 -45% identity to mammalian AQP3 and -9. AQPs belong to the major intrinsic protein family and represent channels for small non-ionic molecules. Both TbAQP1 and TbAQP3 contain two highly conserved NPA motifs within the pore-forming region, whereas TbAQP2 contains NSA and NPS motifs instead, which are only occasionally found in AQPs. For functional characterization, all three proteins were heterologously expressed in yeast and Xenopus oocytes. In the yeast fps1⌬ mutant, TbAQPs suppressed hypoosmosensitivity and rendered cells to a hyper-osmosensitive phenotype, as expected for unregulated glycerol channels. Under iso-and hyperosmotic conditions, these cells constitutively released glycerol, consistent with a glycerol efflux function of TbAQP proteins. TbAQP expression in Xenopus oocytes increased permeability for water, glycerol and, interestingly, dihydroxyacetone. Except for urea, TbAQPs were virtually impermeable for other polyols; only TbAQP3 transported erythritol and ribitol. Thus, TbAQPs represent mainly water/glycerol/dihydroxyacetone channels involved in osmoregulation and glycerol metabolism in T. brucei. This function and especially the so far not investigated transport of dihydroxyacetone may be pivotal for the survival of the parasite survival under non-aerobic or osmotic stress conditions.

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“…Both mentioned opportunities were exploited previously by our group, culminating in the design of a trypanosomatid-selective inhibitor N 6 -(1-naphthalenemethyl)-2′-deoxy-2′-(3-methoxybenzamido)adenosine (6e) 24 with IC 50 values that have been determined to be 25,12, and 6 μM for T. brucei, T. cruzi, and L. mexicana GAPDH's, respectively.…”

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confidence: 99%

Adenosine Analogues as Selective Inhibitors of Glyceraldehyde-3-phosphate Dehydrogenase of Trypanosomatidae via Structure-Based Drug Design

et al. 2001

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In our continuation of the structure-based design of anti-trypanosomatid drugs, parasite-selective adenosine analogues were identified as low micromolar inhibitors of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Crystal structures of Trypanosoma brucei, Trypanosoma cruzi, Leishmania mexicana, and human GAPDH's provided details of how the adenosyl moiety of NAD + interacts with the proteins, and this facilitated the understanding of the relative affinities of a series of adenosine analogues for the various GAPDH's. From exploration of modifications of the naphthalenemethyl and benzamide substituents of a lead compound, N 6 -(1-naphthalenemethyl)-2′-deoxy-2′-(3-methoxybenzamido)adenosine (6e), N 6 -(substituted-naphthalenemethyl)-2′-deoxy-2′-(substituted-benzamido)adenosine analogues were investigated. N 6 -(1-Naphthalenemethyl)-2′-deoxy-2′-(3,5-dimethoxybenzamido)adenosine (6m), N 6 -[1-(3-hydroxy-naphthalene)methyl]-2′-deoxy-2′-(3,5-dimethoxybenzamido)adenosine (7m), N 6 -[1-(3-methoxy-naphthalene)methyl]-2′-deoxy-2′-(3,5-dimethoxybenzamido)adenosine (9m), N 6 -(2-naphthalene-methyl)-2′-deoxy-2′-(3-methoxybenzamido)adenosine (11e), and N 6 -(2-naphthalenemethyl)-2′-deoxy-2′-(3,5-dimethoxybenzamido)adenosine (11m) demonstrated a 2-to 3-fold improvement over 6e and a 7100-to 25000-fold improvement over the adenosine template. IC 50 's of these compounds were in the range 2-12 μM for T. brucei, T. cruzi, and L. mexicana GAPDH's, and these compounds did not inhibit mammalian GAPDH when tested at their solubility limit. To explore more thoroughly the structure-activity relationships of this class of compounds, a library of 240 N 6 -(substituted)-2′-deoxy-2′-(amido)adenosine analogues was generated using parallel solution-phase synthesis with N 6 and C2′ substituents chosen on the basis of computational docking scores. This resulted in the identification of 40 additional compounds that inhibit parasite GAPDH's in the low micromolar range. We also explored adenosine analogues containing 5′-amido substituents and found that 2′,5′-

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Glycolysis in Bloodstream Form Trypanosoma brucei Can Be Understood in Terms of the Kinetics of the Glycolytic Enzymes

Cited by 193 publications

References 46 publications

Depletion of GIM5 Causes Cellular Fragility, a Decreased Glycosome Number, and Reduced Levels of Ether-linked Phospholipids in Trypanosomes

Depletion of GIM5 Causes Cellular Fragility, a Decreased Glycosome Number, and Reduced Levels of Ether-linked Phospholipids in Trypanosomes

Cloning, Heterologous Expression, and Characterization of Three Aquaglyceroporins from Trypanosoma brucei

Adenosine Analogues as Selective Inhibitors of Glyceraldehyde-3-phosphate Dehydrogenase of Trypanosomatidae via Structure-Based Drug Design

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