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Paul A M Michels - One of the best experts on this subject based on the ideXlab platform.

  • Peroxisomes, glyoxysomes and Glycosomes (review).
    Molecular membrane biology, 2020
    Co-Authors: Paul A M Michels, Juliette Moyersoen, Hanane Krazy, Nathalie Galland, Murielle Herman, Véronique Hannaert
    Abstract:

    Peroxisomes, glyoxysomes and Glycosomes are related organelles found in different organisms. The morphology and enzymic content of the different members of this organelle family differ considerably, and may also be highly dependent on the cell's environmental conditions or life cycle. However, all peroxisome-like organelles have in common a number of characteristic enzymes or enzyme systems, notably enzymes dealing with reactive oxygen species. All organelles of the family follow essentially the same route of biogenesis, but with species-specific differences. Sets of proteins called peroxins are involved in different aspects of the formation and proliferation of peroxisomes such as import of proteins in the organellar matrix, insertion of proteins in the membrane, etc. In different eukaryotic lineages these functions are carried out by often--but not always--homologous yet poorly conserved peroxins. The process of biogenesis and the nature of the proteins involved suggest that all members of the peroxisome family evolved from a single organelle in an ancestral eukaryotic cell. This original peroxisome was possibly derived from a cellular membrane system such as the endoplasmic reticulum. Most of the organism-specific functions of the extant organelles have been acquired later in evolution.

  • Structure, Properties, and Function of Glycosomes in Trypanosoma cruzi.
    Frontiers in Cellular and Infection Microbiology, 2020
    Co-Authors: Wilfredo Quinones, Hector Acosta, Melisa Gualdrón-lópez, Camila Silva Gonçalves, Maria Cristina M. Motta, Paul A M Michels
    Abstract:

    Glycosomes are peroxisome-related organelles that have been identified in kinetoplastids and diplonemids. The hallmark of Glycosomes is their harbouring of the majority of the glycolytic enzymes. Our biochemical studies and proteome analysis of Trypanosoma cruzi Glycosomes have located, in addition to enzymes of the glycolytic pathway, enzymes of several other metabolic processes in the organelles. These analyses revealed many aspects in common with Glycosomes from other trypanosomatids as well as features that seem specific for T. cruzi. Their enzyme content indicates that T. cruzi Glycosomes are multifunctional organelles, involved in both several catabolic processes such as glycolysis and anabolic ones. Specifically discussed in this minireview are the cross-talk between glycosomal metabolism and metabolic processes occurring in other cell compartments, and the importance of metabolite translocation systems in the glycosomal membrane to enable the coordination between the spatially separated processes. Possible mechanisms for metabolite translocation across the membrane are suggested by proteins identified in the organelle’s membrane – homologs of the ABC and MCF transporter families – and the presence of channels as inferred previously from the detection of channel-forming proteins in glycosomal membrane preparations from the related parasite T. brucei. Together, these data provide insight in the way in which different parts of T. cruzi metabolism, although uniquely distributed over different compartments, are integrated and regulated. Moreover, this information reveals opportunities for the development of drugs against Chagas disease caused by these parasites and for which currently no adequate treatment is available.

  • Proteomic analysis of Glycosomes from Trypanosoma cruzi epimastigotes
    Molecular and Biochemical Parasitology, 2019
    Co-Authors: Hector Acosta, Paul A M Michels, Juan Luis Concepcion, Ana J Caceres, Melisa Gualdrón-lópez, Richard Burchmore, Christina Naula, Ender Quintero-troconis, Wilfredo Quinones
    Abstract:

    In Trypanosoma cruzi, the causal agent of Chagas disease, the first seven steps of glycolysis are compartmentalized in Glycosomes, which are authentic but specialized peroxisomes. Besides glycolysis, activity of enzymes of other metabolic processes have been reported to be present in Glycosomes, such as β-oxidation of fatty acids, purine salvage, pentose-phosphate pathway, gluconeogenesis and biosynthesis of ether-lipids, isoprenoids, sterols and pyrimidines. In this study, we have purified Glycosomes from T. cruzi epimastigotes, collected the soluble and membrane fractions of these organelles, and separated peripheral and integral membrane proteins by Na2CO3 treatment and osmotic shock. Proteomic analysis was performed on each of these fractions, allowing us to confirm the presence of enzymes involved in various metabolic pathways as well as identify new components of this parasite’s Glycosomes.

  • the kinetic characteristics of human and trypanosomatid phosphofructokinases for the reverse reaction
    Biochemical Journal, 2019
    Co-Authors: Peter M Fernandes, Paul A M Michels, Frederic Bringaud, James Kinkead, I W Mcnae, Malcolm D Walkinshaw
    Abstract:

    Eukaryotic ATP-dependent phosphofructokinases (PFKs) are often considered unidirectional enzymes  catalysing the transfer of a phospho moiety from ATP to fructose 6-phosphate (F6P) to produce ADP and fructose 1,6-bisphosphate (F16BP). The reverse reaction is not generally considered to occur under normal conditions and has never been demonstrated for any eukaryotic ATP-dependent PFKs, though it does occur in PPi-dependent PFKs and has been experimentally shown for bacterial ATP-dependent PFKs. Evidence is provided via two orthogonal assays that all three human PFK isoforms can catalyse the reverse reaction in vitro , allowing determination of kinetic properties. Additionally, the reverse reaction was shown possible for PFKs from three clinically important trypanosomatids; these enzymes are contained within Glycosomes in vivo . This compartmentalisation may facilitate reversal, given the potential for trypanosomatids to have an altered ATP/ADP ratio in Glycosomes compared to the cytosol. The kinetic properties of each trypanosomatid PFK were determined, including the response to natural and artificial modulators of enzyme activity. The possible physiological relevance of the reverse reaction in trypanosomatid and human PFKs is discussed.

  • Biogenesis, maintenance and dynamics of Glycosomes in trypanosomatid parasites
    Biochimica et Biophysica Acta, 2015
    Co-Authors: Jurgen R. Haanstra, Eglys González-marcano, Melisa Gualdrón-lópez, Paul A M Michels
    Abstract:

    Peroxisomes of organisms belonging to the protist group Kinetoplastea, which include trypanosomatid parasites of the genera Trypanosoma and Leishmania, are unique in playing a crucial role in glycolysis and other parts of intermediary metabolism. They sequester the majority of the glycolytic enzymes and hence are called Glycosomes. Their glycosomal enzyme content can vary strongly, particularly quantitatively, between different trypanosomatid species, and within each species during its life cycle. Turnover of Glycosomes by autophagy of redundant ones and biogenesis of a new population of organelles play a pivotal role in the efficient adaptation of the glycosomal metabolic repertoire to the sudden, major nutritional changes encountered during the transitions in their life cycle. The overall mechanism of Glycosome biogenesis is similar to that of peroxisomes in other organisms, but the homologous peroxins involved display low sequence conservation as well as variations in motifs mediating crucial protein-protein interactions in the process. The correct compartmentalisation of enzymes is essential for the regulation of the trypanosomatids' metabolism and consequently for their viability. For Trypanosoma brucei it was shown that Glycosomes also play a crucial role in its life-cycle regulation: a crucial developmental control switch involves the translocation of a protein phosphatase from the cytosol into the organelles. Many glycosomal proteins are differentially phosphorylated in different life-cycle stages, possibly indicative of regulation of enzyme activities as an additional means to adapt the metabolic network to the different environmental conditions encountered.

Wilfredo Quinones - One of the best experts on this subject based on the ideXlab platform.

  • The Glycosome membrane of Trypanosoma cruzi epimastigotes: protein and lipid composition.
    Experimental parasitology, 2020
    Co-Authors: Wilfredo Quinones, Julio A Urbina, Michel Dubourdieu, Juan Luis Concepción
    Abstract:

    Highly purified Glycosomes from Trypanosoma cruzi epimastigotes were obtained by differential centrifugation and isopycnic ultracentrifugation. Glycosomal membranes, produced by carbonate treatment of purified Glycosomes, exhibited about eight main protein bands and eight minor ones. Essentially the same protein pattern was observed in the detergent-rich fraction of a Triton X-114 fractionation of whole Glycosomes, indicating that most of the membrane-bound polypeptides were highly hydrophobic. The orientation of these proteins was studied by in situ labelling followed by limited pronase hydrolysis of intact Glycosomes. Three Glycosome membrane proteins were characterized as peripheral by comparing the protein bands patterns of membrane fractions obtained by different treatments. Noteworthy membrane polypeptides were: (1) a peripheral 75k Da membrane protein, oriented towards the cytosol, which was the most abundant glycosomal membrane protein in exponentially growing epimastigotes but was essentially absent in stationary phase cells; (2) a pair of integral membrane proteins with molecular masses in the range of 85-100 kDa, which were only present in stationary phase cells; (3) a heme-containing 36k Da protein, strongly associated to the membrane, present in both growth phases; (4) a very immunogenic 41k Da integral membrane polypeptide, oriented towards the cytosol. The lipid composition of the glycosomal membranes was also investigated. The distribution of phospholipid species in Glycosomes and glycosomal membranes was very similar to that of whole cells, with phosphatidyl-ethanolamine, phosphatidyl-choline, and phosphatidyl-serine as main components and smaller proportions of sphingomyelin and with phosphatidyl-inositol. On the other hand, Glycosomes were enriched in endogenous sterols (ergosterol, 24-ethyl-5,7,22-cholesta-trien-3beta-ol), and precursors, when compared with whole cells, a finding consistent with the proposal that these organelles are involved in the de novo biosynthesis of sterols in trypanosomatids.

  • Isolation of Glycosomes from Trypanosoma cruzi.
    Methods of Molecular Biology, 2020
    Co-Authors: Hector Acosta, Wilfredo Quinones
    Abstract:

    : Glycosomes are peroxisome-related organelles of trypanosomatids in which the glycolytic and some other metabolic pathways are compartmentalized. We describe here two methods for the purification of Glycosomes from Trypanosoma cruzi for preparative purposes, differential and isopycnic centrifugation. These are two techniques that allow the separation of different cellular compartments based on their different physicochemical characteristics. The first type of centrifugation is a rapid method that does not require large inputs and allows for fractions enriched in specific cell compartments to be obtained. The second type of centrifugation is a more elaborate method, but enables highly purified cellular compartments to be isolated. The success in obtaining these purified, intact organelles critically depends on using an appropriate method for controlled rupture of the cells.

  • Structure, Properties, and Function of Glycosomes in Trypanosoma cruzi.
    Frontiers in Cellular and Infection Microbiology, 2020
    Co-Authors: Wilfredo Quinones, Hector Acosta, Melisa Gualdrón-lópez, Camila Silva Gonçalves, Maria Cristina M. Motta, Paul A M Michels
    Abstract:

    Glycosomes are peroxisome-related organelles that have been identified in kinetoplastids and diplonemids. The hallmark of Glycosomes is their harbouring of the majority of the glycolytic enzymes. Our biochemical studies and proteome analysis of Trypanosoma cruzi Glycosomes have located, in addition to enzymes of the glycolytic pathway, enzymes of several other metabolic processes in the organelles. These analyses revealed many aspects in common with Glycosomes from other trypanosomatids as well as features that seem specific for T. cruzi. Their enzyme content indicates that T. cruzi Glycosomes are multifunctional organelles, involved in both several catabolic processes such as glycolysis and anabolic ones. Specifically discussed in this minireview are the cross-talk between glycosomal metabolism and metabolic processes occurring in other cell compartments, and the importance of metabolite translocation systems in the glycosomal membrane to enable the coordination between the spatially separated processes. Possible mechanisms for metabolite translocation across the membrane are suggested by proteins identified in the organelle’s membrane – homologs of the ABC and MCF transporter families – and the presence of channels as inferred previously from the detection of channel-forming proteins in glycosomal membrane preparations from the related parasite T. brucei. Together, these data provide insight in the way in which different parts of T. cruzi metabolism, although uniquely distributed over different compartments, are integrated and regulated. Moreover, this information reveals opportunities for the development of drugs against Chagas disease caused by these parasites and for which currently no adequate treatment is available.

  • Proteomic analysis of Glycosomes from Trypanosoma cruzi epimastigotes
    Molecular and Biochemical Parasitology, 2019
    Co-Authors: Hector Acosta, Paul A M Michels, Juan Luis Concepcion, Ana J Caceres, Melisa Gualdrón-lópez, Richard Burchmore, Christina Naula, Ender Quintero-troconis, Wilfredo Quinones
    Abstract:

    In Trypanosoma cruzi, the causal agent of Chagas disease, the first seven steps of glycolysis are compartmentalized in Glycosomes, which are authentic but specialized peroxisomes. Besides glycolysis, activity of enzymes of other metabolic processes have been reported to be present in Glycosomes, such as β-oxidation of fatty acids, purine salvage, pentose-phosphate pathway, gluconeogenesis and biosynthesis of ether-lipids, isoprenoids, sterols and pyrimidines. In this study, we have purified Glycosomes from T. cruzi epimastigotes, collected the soluble and membrane fractions of these organelles, and separated peripheral and integral membrane proteins by Na2CO3 treatment and osmotic shock. Proteomic analysis was performed on each of these fractions, allowing us to confirm the presence of enzymes involved in various metabolic pathways as well as identify new components of this parasite’s Glycosomes.

  • subcellular localization of glycolytic enzymes and characterization of intermediary metabolism of trypanosoma rangeli
    Molecular and Biochemical Parasitology, 2017
    Co-Authors: Rocio Rondonmercado, Wilfredo Quinones, Hector Acosta, Ana J Caceres, Juan Luis Concepcion
    Abstract:

    Trypanosoma rangeli is a hemoflagellate protist that infects wild and domestic mammals as well as humans in Central and South America. Although this parasite is not pathogenic for human, it is being studied because it shares with Trypanosoma cruzi, the etiological agent of Chagas' disease, biological characteristics, geographic distribution, vectors and vertebrate hosts. Several metabolic studies have been performed with T. cruzi epimastigotes, however little is known about the metabolism of T. rangeli. In this work we present the subcellular distribution of the T. rangeli enzymes responsible for the conversion of glucose to pyruvate, as determined by epifluorescense immunomicroscopy and subcellular fractionation involving either selective membrane permeabilization with digitonin or differential and isopycnic centrifugation. We found that in T. rangeli epimastigotes the first six enzymes of the glycolytic pathway, involved in the conversion of glucose to 1,3-bisphosphoglycerate are located within Glycosomes, while the last four steps occur in the cytosol. In contrast with T. cruzi, where three isoenzymes (one cytosolic and two glycosomal) of phosphoglycerate kinase are expressed simultaneously, only one enzyme with this activity is detected in T. rangeli epimastigotes, in the cytosol. Consistent with this latter result, we found enzymes involved in auxiliary pathways to glycolysis needed to maintain adenine nucleotide and redox balances within Glycosomes such as phosphoenolpyruvate carboxykinase, malate dehydrogenase, fumarate reductase, pyruvate phosphate dikinase and glycerol-3-phosphate dehydrogenase. Glucokinase, galactokinase and the first enzyme of the pentose-phosphate pathway, glucose-6-phosphate dehydrogenase, were also located inside Glycosomes. Furthermore, we demonstrate that T. rangeli epimastigotes growing in LIT medium only consume glucose and do not excrete ammonium; moreover, they are unable to survive in partially-depleted glucose medium. The velocity of glucose consumption is about 40% higher than that of procyclic Trypanosoma brucei, and four times faster than by T. cruzi epimastigotes under the same culture conditions.

Juan Luis Concepcion - One of the best experts on this subject based on the ideXlab platform.

  • Proteomic analysis of Glycosomes from Trypanosoma cruzi epimastigotes
    Molecular and Biochemical Parasitology, 2019
    Co-Authors: Hector Acosta, Paul A M Michels, Juan Luis Concepcion, Ana J Caceres, Melisa Gualdrón-lópez, Richard Burchmore, Christina Naula, Ender Quintero-troconis, Wilfredo Quinones
    Abstract:

    In Trypanosoma cruzi, the causal agent of Chagas disease, the first seven steps of glycolysis are compartmentalized in Glycosomes, which are authentic but specialized peroxisomes. Besides glycolysis, activity of enzymes of other metabolic processes have been reported to be present in Glycosomes, such as β-oxidation of fatty acids, purine salvage, pentose-phosphate pathway, gluconeogenesis and biosynthesis of ether-lipids, isoprenoids, sterols and pyrimidines. In this study, we have purified Glycosomes from T. cruzi epimastigotes, collected the soluble and membrane fractions of these organelles, and separated peripheral and integral membrane proteins by Na2CO3 treatment and osmotic shock. Proteomic analysis was performed on each of these fractions, allowing us to confirm the presence of enzymes involved in various metabolic pathways as well as identify new components of this parasite’s Glycosomes.

  • subcellular localization of glycolytic enzymes and characterization of intermediary metabolism of trypanosoma rangeli
    Molecular and Biochemical Parasitology, 2017
    Co-Authors: Rocio Rondonmercado, Wilfredo Quinones, Hector Acosta, Ana J Caceres, Juan Luis Concepcion
    Abstract:

    Trypanosoma rangeli is a hemoflagellate protist that infects wild and domestic mammals as well as humans in Central and South America. Although this parasite is not pathogenic for human, it is being studied because it shares with Trypanosoma cruzi, the etiological agent of Chagas' disease, biological characteristics, geographic distribution, vectors and vertebrate hosts. Several metabolic studies have been performed with T. cruzi epimastigotes, however little is known about the metabolism of T. rangeli. In this work we present the subcellular distribution of the T. rangeli enzymes responsible for the conversion of glucose to pyruvate, as determined by epifluorescense immunomicroscopy and subcellular fractionation involving either selective membrane permeabilization with digitonin or differential and isopycnic centrifugation. We found that in T. rangeli epimastigotes the first six enzymes of the glycolytic pathway, involved in the conversion of glucose to 1,3-bisphosphoglycerate are located within Glycosomes, while the last four steps occur in the cytosol. In contrast with T. cruzi, where three isoenzymes (one cytosolic and two glycosomal) of phosphoglycerate kinase are expressed simultaneously, only one enzyme with this activity is detected in T. rangeli epimastigotes, in the cytosol. Consistent with this latter result, we found enzymes involved in auxiliary pathways to glycolysis needed to maintain adenine nucleotide and redox balances within Glycosomes such as phosphoenolpyruvate carboxykinase, malate dehydrogenase, fumarate reductase, pyruvate phosphate dikinase and glycerol-3-phosphate dehydrogenase. Glucokinase, galactokinase and the first enzyme of the pentose-phosphate pathway, glucose-6-phosphate dehydrogenase, were also located inside Glycosomes. Furthermore, we demonstrate that T. rangeli epimastigotes growing in LIT medium only consume glucose and do not excrete ammonium; moreover, they are unable to survive in partially-depleted glucose medium. The velocity of glucose consumption is about 40% higher than that of procyclic Trypanosoma brucei, and four times faster than by T. cruzi epimastigotes under the same culture conditions.

  • Trypanosomatid Diseases: Molecular Routes to Drug Discovery - Function of Glycosomes in the Metabolism of Trypanosomatid Parasites and the Promise of Glycosomal Proteins as Drug Targets
    Trypanosomatid Diseases, 2013
    Co-Authors: Melisa Gualdrón-lópez, Paul A M Michels, Wilfredo Quinones, Ana J Caceres, Luisana Avilán, Juan Luis Concepcion
    Abstract:

    Trypanosomatids have the unique feature of compartmentalizing the major part of the glycolytic pathway inside peroxisome-related organelles called Glycosomes. However, these organelles also contain enzymes of several other important pathways involved in both catabolic and anabolic processes. The enzyme content and the metabolic role of Glycosomes differ between trypanosomatid species and between their life cycle stages. Several of the glycosomal pathways have been shown to be important for the viability, pathogenicity, and/or virulence of different trypanosomatid parasites. Additionally, the correct compartmentalization of glycosomal enzymes inside the organelles appeared to be vital for these pathogens. Therefore, many of these enzymes, as well as the proteins involved in the translocation of metabolites across the glycosomal membrane and peroxins (PEXs), proteins responsible for the biogenesis of Glycosomes, are candidate drug targets. Glycosomal enzymes and PEX proteins of Trypanosoma brucei, T. cruzi, and Leishmania spp. are being studied, and compounds that interfere with their functioning are being developed for use as lead drugs against the diseases caused by these parasites. Potent, selective inhibitors of several enzymes have been obtained that exert trypanocidal activity on parasites cultured in vitro and have no or only little effect on growth of human cells. In addition, some compounds showed anti-parasite activity in experimentally infected animals.

  • pyruvate phosphate dikinase and pyrophosphate metabolism in the Glycosome of trypanosoma cruzi epimastigotes
    Comparative Biochemistry and Physiology B, 2004
    Co-Authors: Hector Acosta, Frederic Bringaud, Wilfredo Quinones, Michel Dubourdieu, Ana J Caceres, Juan Luis Concepcion
    Abstract:

    Abstract Pyruvate phosphate dikinase (PPDK) was recently reported in trypanosomatids, but its metabolic function is not yet known. The present work deals with the cellular localization and the function of the Trypanosoma cruzi enzyme. First, we show by digitonin titration and cell fractionation that the enzyme was essentially present in the Glycosome matrix of the epimastigote form. Second, we address the issue of the direction of the reaction inside the Glycosome for one part, our bibliographic survey evidenced a quite exergonic ΔG°′ (at least −5.2 kcal/mol at neutral pH and physiologic ionic strength); for another part, no pyrophosphatase (PPase) could be detected in fractions corresponding to the Glycosomes; therefore, glycosomal PPDK likely works in the direction of pyruvate production. Third, we address the issue of the origin of the glycosomal pyrophosphate (PPi): several synthetic pathways known to produce PPi are already considered to be glycosomal. This work also indicates the presence of an NADP+-dependent β-oxidation of palmitoyl-CoA in the Glycosome. Several pyruvate-consuming activities, in particular alanine dehydrogenase (ADH) and pyruvate carboxylase (PC), were detected in the glycosomal fraction. PPDK appears therefore as a central enzyme in the metabolism of the Glycosome of T. cruzi by providing a link between glycolysis, fatty acid oxidation and biosynthetic PPi-producing pathways. Indeed, PPDK seems to replace pyrophosphatase in its classical thermodynamic role of displacing the equilibrium of PPi-producing reactions, as well as in its role of eliminating the toxic PPi.

  • the Glycosome membrane of trypanosoma cruzi epimastigotes protein and lipid composition
    Experimental Parasitology, 2004
    Co-Authors: Wilfredo Quinones, Julio A Urbina, Michel Dubourdieu, Juan Luis Concepcion
    Abstract:

    Abstract Highly purified Glycosomes from Trypanosoma cruzi epimastigotes were obtained by differential centrifugation and isopycnic ultracentrifugation. Glycosomal membranes, produced by carbonate treatment of purified Glycosomes, exhibited about eight main protein bands and eight minor ones. Essentially the same protein pattern was observed in the detergent-rich fraction of a Triton X-114 fractionation of whole Glycosomes, indicating that most of the membrane-bound polypeptides were highly hydrophobic. The orientation of these proteins was studied by in situ labelling followed by limited pronase hydrolysis of intact Glycosomes. Three Glycosome membrane proteins were characterized as peripheral by comparing the protein bands patterns of membrane fractions obtained by different treatments. Noteworthy membrane polypeptides were: (1) a peripheral 75 kDa membrane protein, oriented towards the cytosol, which was the most abundant glycosomal membrane protein in exponentially growing epimastigotes but was essentially absent in stationary phase cells; (2) a pair of integral membrane proteins with molecular masses in the range of 85–100 kDa, which were only present in stationary phase cells; (3) a heme-containing 36 kDa protein, strongly associated to the membrane, present in both growth phases; (4) a very immunogenic 41 kDa integral membrane polypeptide, oriented towards the cytosol. The lipid composition of the glycosomal membranes was also investigated. The distribution of phospholipid species in Glycosomes and glycosomal membranes was very similar to that of whole cells, with phosphatidyl-ethanolamine, phosphatidyl-choline, and phosphatidyl-serine as main components and smaller proportions of sphingomyelin and with phosphatidyl-inositol. On the other hand, Glycosomes were enriched in endogenous sterols (ergosterol, 24-ethyl-5,7,22-cholesta-trien-3β-ol), and precursors, when compared with whole cells, a finding consistent with the proposal that these organelles are involved in the de novo biosynthesis of sterols in trypanosomatids. Index Descriptors and Abbreviations: Glycosome, Glycosome membrane proteins, Trypanosoma cruzi , epimastigote, phospholipids, neutral lipids, sterols; LIT, liver infusion-tryptose; TMBZ, 3,3 ′ ,5,5 ′ -tetramethylbenzamidine; GMPs, Glycosome membrane proteins; HK, hexokinase, EC 2.7.1.1 ; PGI, phosphoglucose isomerase, EC 5.3.1.9 ; MDH, malate dehydrogenase, EC 1.1.1.37 ; GDH, α-glycerol-3-phosphate dehydrogenase, EC 1.1.1.8 ; PK, pyruvate kinase, EC 2.7.1.40 ; PEPCK, phosphoenolpyruvate carboxykinase, EC 4.1.1.4.9 ; PGK, 3-phosphoglycerate kinase, EC 2.7.2.3 ; PPDK, phosphate pyruvate dikinase, EC 2.7.9.1 ; NL, neutral lipids; PL, phospholipids; TCA, trichloracetic acid

Meredith Morris - One of the best experts on this subject based on the ideXlab platform.

  • trypanosoma brucei pex13 2 is an accessory peroxin that functions in the import of peroxisome targeting sequence type 2 proteins and localizes to subdomains of the Glycosome
    mSphere, 2020
    Co-Authors: Logan P Crowe, Christina L Wilkinson, Kathleen R Nicholson, Meredith Morris
    Abstract:

    ABSTRACT Kinetoplastid parasites, including Trypanosoma brucei, Trypanosoma cruzi, and Leishmania, harbor unique organelles known as Glycosomes, which are evolutionarily related to peroxisomes. Glycosome/peroxisome biogenesis is mediated by proteins called peroxins that facilitate organelle formation, proliferation, and degradation and import of proteins housed therein. Import of matrix proteins occurs via one of two pathways that are dictated by their peroxisome targeting sequence (PTS). In PTS1 import, a C-terminal tripeptide sequence, most commonly SKL, is recognized by the soluble receptor Pex5. In PTS2 import, a less conserved N-terminal sequence is recognized by Pex7. The soluble receptors deliver their cargo to the import channel consisting minimally of Pex13 and Pex14. While much of the import process is conserved, kinetoplastids are the only organisms to have two Pex13s, Pex13.1 and Pex13.2. It is unclear why trypanosomes require two Pex13s when one is sufficient for most eukaryotes. To interrogate the role of Pex13.2, we have employed biochemical approaches to partially resolve the composition of the Pex13/Pex14 import complexes in T. brucei and characterized Glycosome morphology and protein import in Pex13.2-deficient parasites. Here, we show that Pex13.2 is an integral Glycosome membrane protein that interacts with Pex13.1 and Pex14. The N terminus of Pex13.2 faces the cytoplasmic side of the membrane, where it can facilitate interactions required for protein import. Two-dimensional gel electrophoresis revealed three Glycosome membrane complexes containing combinations of Pex13.1, Pex13.2, and Pex14. The silencing of Pex13.2 resulted in parasites with fewer, larger Glycosomes and disrupted Glycosome protein import, suggesting the protein is involved in Glycosome biogenesis as well as protein import. Furthermore, superresolution microscopy demonstrated that Pex13.2 localizes to discrete foci in the Glycosome periphery, indicating that the Glycosome periphery is not homogenous. IMPORTANCETrypanosoma brucei causes human African trypanosomiasis and a wasting disease called Nagana in livestock. Current treatments are expensive, toxic, and difficult to administer. Because of this, the search for new drug targets is essential. T. brucei has Glycosomes that are essential to parasite survival; however, our ability to target them in drug development is hindered by our lack of understanding about how these organelles are formed and maintained. This work forwards our understanding of how the parasite-specific protein Pex13.2 functions in Glycosome protein import and lays the foundation for future studies focused on blocking Pex13.2 function, which would be lethal to bloodstream-form parasites that reside in the mammalian bloodstream.

  • trypanosoma brucei pex13 2 is an accessory peroxin that functions in the import of pts2 proteins and localizes to subdomains of the Glycosome
    bioRxiv, 2018
    Co-Authors: Logan P Crowe, Christina L Wilkinson, Kathleen R Nicholson, Meredith Morris
    Abstract:

    Kinetoplastid parasites including Trypanosoma brucei, Trypanosoma cruzi and Leishmania harbor unique organelles known as Glycosomes, which are evolutionarily related to peroxisomes. Glycosome/peroxisome biogenesis is mediated by proteins called peroxins that facilitate organelle formation, proliferation and degradation, and import of proteins housed therein. Import of matrix proteins occurs via one of two pathways that are dictated by their peroxisome targeting sequence (PTS). In PTS1 import, a C-terminal tripeptide sequence, most commonly SKL, is recognized by the soluble receptor Pex5. In PTS2 import, a less conserved N-terminal sequence is recognized by Pex7. The soluble receptors deliver their cargo to the import channel consisting minimally of Pex13 and Pex14. While much of the import process is conserved, kinetoplastids are the only organisms to have two Pex13s, TbPex13.1 and TbPex13.2. In previous studies, GFP-tagged TbPex13.1 localized to Glycosomes and silencing either protein in the stage of the parasite that lives in the mammalian bloodstream impaired Glycosome protein import and slowed parasite growth. While these findings suggest Pex13s are involved in protein import, the mechanisms by which they function are unknown and it is unclear why kinetoplastids would require two Pex13s. In this work, we demonstrate that TbPex13.2 is associated with the Glycosome membrane with its N-terminus facing the cytoplasm. Super-resolution microscopy reveals that TbPex13.2 localizes to a few (1-3) foci per Glycosome and import of PTS2 proteins was disrupted in TbPex13.2-deficient cells suggesting it may be an accessory factor for PTS2 import.

  • Glycosome biogenesis in trypanosomes and the de novo dilemma
    PLOS Neglected Tropical Diseases, 2017
    Co-Authors: Sarah Bauer, Meredith Morris
    Abstract:

    Trypanosomatid parasites, including Trypanosoma and Leishmania, are the causative agents of lethal diseases threatening millions of people around the world. These organisms compartmentalize glycolysis in essential, specialized peroxisomes called Glycosomes. Peroxisome proliferation can occur through growth and division of existing organelles and de novo biogenesis from the endoplasmic reticulum. The level that each pathway contributes is debated. Current evidence supports the concerted contribution of both mechanisms in an equilibrium that can vary depending on environmental conditions and metabolic requirements of the cell. Homologs of a number of peroxins, the proteins involved in peroxisome biogenesis and matrix protein import, have been identified in T. brucei. Based on these findings, it is widely accepted that Glycosomes proliferate through growth and division of existing organelles; however, to our knowledge, a de novo mechanism of biogenesis has not been directly demonstrated. Here, we review recent findings that provide support for the existence of an endoplasmic reticulum (ER)-derived de novo pathway of Glycosome biogenesis in T. brucei. Two studies recently identified PEX13.1, a peroxin involved in matrix protein import, in the ER of procyclic form T. brucei. In other eukaryotes, peroxins including PEX13 have been found in the ER of cells undergoing de novo biogenesis of peroxisomes. In addition, PEX16 and PEX19 have been characterized in T. brucei, both of which are important for de novo biogenesis in other eukaryotes. Because Glycosomes are rapidly remodeled via autophagy during life cycle differentiation, de novo biogenesis could provide a method of restoring Glycosome populations following turnover. Together, the findings we summarize provide support for the hypothesis that Glycosome proliferation occurs through growth and division of pre-existing organelles and de novo biogenesis of new organelles from the ER and that the level each mechanism contributes is influenced by glucose availability.

  • pH regulation in Glycosomes of procyclic form Trypanosoma brucei
    Journal of Biological Chemistry, 2017
    Co-Authors: Charles Voyton, Meredith Morris, James C Morris, P. Christine Ackroyd, Kenneth A. Christensen
    Abstract:

    Abstract Here 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.

  • FRET Cytometric Method for High Throughput Screening of Potential Metabolic Inhibitors in Trypanosoma brucei
    The FASEB Journal, 2016
    Co-Authors: Charles Voyton, Meredith Morris, James C Morris, P. Christine Ackroyd, Kenneth A. Christensen
    Abstract:

    The infectious form of Trypanosoma brucei survives in the mammalian host by converting blood glucose into ATP in a specialized peroxisome-like organelle called the Glycosome. Compartmentalization o...

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  • examination of the mode of action of the almiramide family of natural products against the kinetoplastid parasite trypanosoma brucei
    Journal of Natural Products, 2013
    Co-Authors: Laura M Sanchez, Christine Clayton, Giselle M Knudsen, Claudia Helbig, Geraldine De Muylder, Samantha J Mascuch, Zachary B Mackey, Lena Gerwick, James H Mckerrow, Roger G Linington
    Abstract:

    Almiramide C is a marine natural product with low micromolar activity against Leishmania donovani, the causative agent of leishmaniasis. We have now shown that almiramide C is also active against the related parasite Trypanosoma brucei, the causative agent of human African trypanosomiasis. A series of activity-based probes have been synthesized to explore both the molecular target of this compound series in T. brucei lysates and site localization through epifluorescence microscopy. These target identification studies indicate that the almiramides likely perturb glycosomal function through disruption of membrane assembly machinery. Glycosomes, which are organelles specific to kinetoplastid parasites, house the first seven steps of glycolysis and have been shown to be essential for parasite survival in the bloodstream stage. There are currently no reported small-molecule disruptors of Glycosome function, making the almiramides unique molecular probes for this understudied parasite-specific organelle. Additi...

  • depletion of gim5 causes cellular fragility a decreased Glycosome number and reduced levels of ether linked phospholipids in trypanosomes
    Journal of Biological Chemistry, 2003
    Co-Authors: Frank Voncken, Jaap J Van Hellemond, Iris Pfisterer, Alexander G Maier, Stephan Hillmer, Christine Clayton
    Abstract:

    Abstract 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.

  • compartmentation of phosphoglycerate kinase in trypanosoma brucei plays a critical role in parasite energy metabolism
    Proceedings of the National Academy of Sciences of the United States of America, 1998
    Co-Authors: Judith Blattner, Sandra Helfert, Paul A M Michels, Christine Clayton
    Abstract:

    African trypanosomes compartmentalize glycolysis in a microbody, the Glycosome. When growing in the mammalian bloodstream, trypanosomes contain only a rudimentary mitochondrion, and the first seven glycolytic enzymes, including phosphoglycerate kinase, are located in the Glycosome. Procyclic trypanosomes, growing in the gut of tsetse flies, possess a fully developed mitochondrion that is active in oxidative phosphorylation. The first six glycolytic enzymes are still glycosomal, but phosphoglycerate kinase is now found in the cytosol. We demonstrate here that bloodstream trypanosomes are killed by expression of cytosolic phosphoglycerate kinase. The toxicity depends on both enzyme activity and cytosolic location. One possible explanation is that cytosolic phosphoglycerate kinase creates an ATP-generating shunt in the cytosol, thus preventing full ATP regeneration in the Glycosome and ultimately inhibiting the first, ATP-consuming, steps of glycolysis.

  • Elongation and clustering of Glycosomes in Trypanosoma brucei overexpressing the glycosomal Pex11p
    The EMBO Journal, 1998
    Co-Authors: Patrick Lorenz, Alexander G Maier, Eveline Baumgart, Ralf Erdmann, Christine Clayton
    Abstract:

    Kinetoplastid protozoa confine large parts of glycolysis within Glycosomes, which are microbodies related to peroxisomes. We cloned the gene encoding the second most abundant integral membrane protein of Trypanosoma brucei Glycosomes. The 24 kDa protein is very basic and hydrophobic, with two predicted transmembrane domains. It is targeted to peroxisomes when expressed in mammalian cells and yeast. The protein is a functional homologue of Pex11p from Saccharomyces cerevisiae: pex11Delta mutants, which are defective in peroxisome proliferation, can be complemented by the trypanosome gene. Sequence conservation is significant in the N- and C-terminal domains of all putative Pex11p homologues known, from trypanosomes, yeasts and mammals. Several lines of evidence indicate that these domains are oriented towards the cytosol. TbPex11p can form homodimers, like its yeast counterpart. The TbPEX11 gene is essential in trypanosomes. Inducible overexpression of the protein in T.brucei bloodstream forms causes growth arrest, the globular Glycosomes being transformed to clusters of long tubules filling significant proportions of the cytoplasm. Reduced expression results in trypanosomes with fewer, but larger, organelles.

  • IMPORT OF A DHFR HYBRID PROTEIN INTO GlycosomeS IN VIVO IS NOT INHIBITED BY THE FOLATE-ANALOGUE AMINOPTERIN
    Journal of Cell Biology, 1996
    Co-Authors: T Häusler, Y D Stierhof, Elizabeth Wirtz, Christine Clayton
    Abstract:

    Dihydrofolate reductase fusion proteins have been widely used to study conformational properties of polypeptides translocated across membranes. We have studied the import of dihydrofolate reductase fusion proteins into Glycosomes and mitochondria of Trypanosoma brucei. As signal sequences we used the last 22 carboxy-terminal amino acids of glycosomal phosphoglycerate kinase for Glycosomes, and the cleavable presequences of yeast cytochrome b2 or cytochrome oxidase subunit IV for mitochondria. Upon addition of aminopterin, a folate analogue that stabilizes the dihydrofolate reductase moiety, import of the fusion protein targeted to Glycosomes was not inhibited, although the results of protease protection assays showed that the fusion protein could bind the drug. Under the same conditions, import of a DHFR fusion protein targeted to mitochondria was inhibited by aminopterin. When DHFR fusion proteins targeted simultaneously to both Glycosomes and mitochondria were expressed, import into mitochondria was inhibited by aminopterin, whereas uptake of the same proteins into Glycosomes was either unaffected or slightly increased. These findings suggest that the Glycosomes possess either a strong unfolding activity or an unusually large or flexible translocation channel.