Glycerol 3-Phosphate Alters Trypanosoma brucei Hexokinase Activity in Response to Environmental Change

Dodson, Heidi; Morris, Meredith T.; Morris, James C.

doi:10.1074/jbc.m111.235705

Cited by 18 publications

(29 citation statements)

References 29 publications

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“…In this cell line, PF-PTS2-FP (previously named 29-13-pXS2-Aldo-PTS-eYFP), constitutively expressed PTS2-FP is targeted to glycosomes as indicated by fluorescence microscopy (Fig. 1A) (20).…”

Section: Resultsmentioning

confidence: 99%

“…T. brucei 29-13 procyclic-form parasites, which express T7 RNA polymerase as well as the tetracycline repressor, were maintained in SDM-79 as described previously (19). PF-PTS2-FP (previously named pXS2-Aldo-PTS-eYFP [20]) and PF-FP were maintained in SDM-79 containing G418 (15 g/ml), hygromycin (50 g/ml), and blasticidin (15 g/ml). Clonal cell lines were obtained by limiting dilution (ϳ0.33 parasite/well) into 96-well plates.…”

Section: Methodsmentioning

confidence: 99%

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Environmentally Regulated Glycosome Protein Composition in the African Trypanosome

Bauer

Morris

2013

Eukaryot Cell

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Trypanosomes compartmentalize many metabolic enzymes in glycosomes, peroxisome-related microbodies that are essential to parasite survival. While it is understood that these dynamic organelles undergo profound changes in protein composition throughout life cycle differentiation, the adaptations that occur in response to changes in environmental conditions are less appreciated. We have adopted a fluorescent-organelle reporter system in procyclic Trypanosoma brucei by expressing a fluorescent protein (FP) fused to a glycosomal targeting sequence (peroxisome-targeting sequence 2 [PTS2]). In these cell lines, PTS2-FP is localized within import-competent glycosomes, and organelle composition can be analyzed by microscopy and flow cytometry. Using this reporter system, we have characterized parasite populations that differ in their glycosome composition. In glucoserich medium, two parasite populations are observed; one population harbors glycosomes bearing the full repertoire of glycosome proteins, while the other parasite population contains glycosomes that lack the usual glycosome-resident proteins but do contain the glycosome membrane protein TbPEX11. Interestingly, these cells lack TbPEX13, a protein essential for the import of proteins into the glycosome. This bimodal distribution is lost in low-glucose medium. Furthermore, we have demonstrated that changes in environmental conditions trigger changes in glycosome protein composition. These findings demonstrate a level of procyclic glycosome diversity heretofore unappreciated and offer a system by which glycosome dynamics can be studied in live cells. This work adds to our growing understanding of how the regulation of glycosome composition relates to environmental sensing.T rypanosoma brucei, the causative agent of human African trypanosomiasis, has a complex life cycle, with developmental stages in the bloodstream of the mammalian host and the tsetse fly vector. Each host provides a distinct environment in which the parasites must survive. Bloodstream-form (BSF) parasites are bathed in glucose and generate ATP exclusively by glycolysis. While in the tsetse fly, the procyclic-form (PF) parasites experience a drop in glucose levels with a concomitant increase in the availability of amino acids (namely, proline). Under these conditions, the parasite adapts its metabolism, generating ATP from both glycolysis and amino acid metabolism (1).In trypanosomes, many of the enzymes involved in glycolysis are contained within membrane-bounded organelles called glycosomes (reviewed in references 2, 3, and 4). Similarities between the metabolic activities and the matrix protein import machineries of glycosomes and peroxisomes indicate an evolutionary relationship between the two organelles. In contrast to peroxisomes, however, glycosomes are essential, making mechanisms of glycosome biogenesis and maintenance attractive drug targets.Glycosome dynamics are governed by a number of processes, including organelle biogenesis, protein import, and changes in protein composition. In t...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Environmentally Regulated Glycosome Protein Composition in the African Trypanosome

Bauer

Morris

2013

Eukaryot Cell

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“…To continue to synthesize ATP could be damaging to the host cell, either directly as a consequence of usurping host glucose or by depletion of host purines required for ATP synthesis, since the parasite cannot synthesize purines and must salvage them from the host. The finding that ATP is a regulator of HK activity is not novel among HKs (21), though its role in the malaria parasite remains unclear.…”

Section: Discussionmentioning

confidence: 99%

Interrogating a Hexokinase-Selected Small-Molecule Library for Inhibitors of Plasmodium falciparum Hexokinase

Harris

Walker

Drew

et al. 2013

Antimicrob Agents Chemother

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c Parasites in the genus Plasmodium cause disease throughout the tropic and subtropical regions of the world. P. falciparum, one of the deadliest species of the parasite, relies on glycolysis for the generation of ATP while it inhabits the mammalian red blood cell. The first step in glycolysis is catalyzed by hexokinase (HK). While the 55.3-kDa P. falciparum HK (PfHK) shares several biochemical characteristics with mammalian HKs, including being inhibited by its products, it has limited amino acid identity (ϳ26%) to the human HKs, suggesting that enzyme-specific therapeutics could be generated. To that end, interrogation of a selected small-molecule library of HK inhibitors has identified a class of PfHK inhibitors, isobenzothiazolinones, some of which have 50% inhibitory concentrations (IC 50 s) of <1 M. Inhibition was reversible by dilution but not by treatment with a reducing agent, suggesting that the basis for enzyme inactivation was not covalent association with the inhibitor. Lastly, six of these compounds and the related molecule ebselen inhibited P. falciparum growth in vitro (50% effective concentration [EC 50 ] of >0.6 and <6.8 M). These findings suggest that the chemotypes identified here could represent leads for future development of therapeutics against P. falciparum.

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“…Compartmentalization of glycolytic enzymes within the glycosome has been suggested as the primary means of regulating glycolysis (3,4). In addition to compartmentalization, glycosomal pH has been shown to affect or modulate activity of key glycolytic enzymes (2).…”

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confidence: 99%

“…For example, T. brucei residing in the tsetse fly (the procyclic form, PF) 2 can metabolize both glucose and amino acids (primarily proline), whereas only glucose is used while in the mammalian host (2). Compartmentalization of glycolytic enzymes within the glycosome has been suggested as the primary means of regulating glycolysis (3,4).…”

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pH regulation in glycosomes of procyclic form Trypanosoma brucei

Lin

Voyton

Morris

et al. 2017

Journal of Biological Chemistry

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Edited by Thomas SöllnerHere we report the use of a fluorescein-tagged peroxisomal targeting sequence peptide (F-PTS1, acetyl-C{K(FITC)} GGAKL) for investigating pH regulation of glycosomes in live procyclic form Trypanosoma brucei. When added to cells, this fluorescent peptide is internalized within vesicular structures, including glycosomes, and can be visualized after 30 -60 min. Using F-PTS1 we are able to observe the pH conditions inside glycosomes in response to starvation conditions. Previous studies have shown that in the absence of glucose, the glycosome exhibits mild acidification from pH 7.4 ؎ 0.2 to 6.8 ؎ 0.2. Our results suggest that this response occurs under proline starvation as well. This pH regulation is found to be independent from cytosolic pH and requires a source of Na ؉ ions. Glycosomes were also observed to be more resistant to external pH changes than the cytosol; placement of cells in acidic buffers (pH 5) reduced the pH of the cytosol by 0.8 ؎ 0.1 pH units, whereas glycosomal pH decreases by 0.5 ؎ 0.1 pH units. This observation suggests that regulation of glycosomal pH is different and independent from cytosolic pH regulation. Furthermore, pH regulation is likely to work by an active process, because cells depleted of ATP with 2-deoxyglucose and sodium azide were unable to properly regulate pH. Finally, inhibitor studies with bafilomycin and EIPA suggest that both V-ATPases and Na ؉ /H ؉ exchangers are required for glycosomal pH regulation.

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Glycerol 3-Phosphate Alters Trypanosoma brucei Hexokinase Activity in Response to Environmental Change

Cited by 18 publications

References 29 publications

Environmentally Regulated Glycosome Protein Composition in the African Trypanosome

Environmentally Regulated Glycosome Protein Composition in the African Trypanosome

Interrogating a Hexokinase-Selected Small-Molecule Library for Inhibitors of Plasmodium falciparum Hexokinase

pH regulation in glycosomes of procyclic form Trypanosoma brucei

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