RESUMO
Per-ARNT-Sim (PAS) domains constitute a family of domains present in a wide variety of prokaryotic and eukaryotic organisms. They form part of the structure of various proteins involved in diverse cellular processes. Regulation of enzymatic activity and adaptation to environmental conditions, by binding small ligands, are the main functions attributed to PAS-containing proteins. Recently, genes for a diverse set of proteins with a PAS domain were identified in the genomes of several protists belonging to the group of kinetoplastids, however, until now few of these proteins have been characterized. In this work, we characterize a phosphoglycerate kinase containing a PAS domain present in Trypanosoma cruzi (TcPAS-PGK). This PGK isoform is an active enzyme of 58 kDa with a PAS domain located at its N-terminal end. We identified the protein's localization within glycosomes of the epimastigote form of the parasite by differential centrifugation and selective permeabilization of its membranes with digitonin, as well as in an enriched mitochondrial fraction. Heterologous expression systems were developed for the protein with the N-terminal PAS domain (PAS-PGKc) and without it (PAS-PGKt), and the substrate affinities of both forms of the protein were determined. The enzyme does not exhibit standard Michaelis-Menten kinetics. When evaluating the dependence of the specific activity of the recombinant PAS-PGK on the concentration of its substrates 3-phosphoglycerate (3PGA) and ATP, two peaks of maximal activity were found for the complete enzyme with the PAS domain and a single peak for the enzyme without the domain. Km values measured for 3PGA were 219 ± 26 and 8.8 ± 1.3 µM, and for ATP 291 ± 15 and 38 ± 2.2 µM, for the first peak of PAS-PGKc and for PAS-PGKt, respectively, whereas for the second PAS-PGKc peak values of approximately 1.1-1.2 mM were estimated for both substrates. Both recombinant proteins show inhibition by high concentrations of their substrates, ATP and 3PGA. The presence of hemin and FAD exerts a stimulatory effect on PAS-PGKc, increasing the specific activity by up to 55%. This stimulation is not observed in the absence of the PAS domain. It strongly suggests that the PAS domain has an important function in vivo in T. cruzi in the modulation of the catalytic activity of this PGK isoform. In addition, the PAS-PGK through its PAS and PGK domains could act as a sensor for intracellular conditions in the parasite to adjust its intermediary metabolism.
Assuntos
Doença de Chagas , Trypanosoma cruzi , Humanos , Fosfoglicerato Quinase/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Trifosfato de Adenosina/metabolismoRESUMO
Trypanosoma cruzi, the causative agent of Chagas' disease, belongs to the Trypanosomatidae family. The parasite undergoes multiple morphological and metabolic changes during its life cycle, in which it can use both glucose and amino acids as carbon and energy sources. The glycolytic pathway is peculiar in that its first six or seven steps are compartmentalized in glycosomes, and has a two-branched auxiliary glycosomal system functioning beyond the intermediate phosphoenolpyruvate (PEP) that is also used in the cytosol as substrate by pyruvate kinase. The pyruvate phosphate dikinase (PPDK) is the first enzyme of one branch, converting PEP, PPi, and AMP into pyruvate, Pi, and ATP. Here we present a kinetic study of PPDK from T. cruzi that reveals its hysteretic behavior. The length of the lag phase, and therefore the time for reaching higher specific activity values is affected by the concentration of the enzyme, the presence of hydrogen ions and the concentrations of the enzyme's substrates. Additionally, the formation of a more active PPDK with more complex structure is promoted by it substrates and the cation ammonium, indicating that this enzyme equilibrates between the monomeric (less active) and a more complex (more active) form depending on the medium. These results confirm the hysteretic behavior of PPDK and are suggestive for its functioning as a regulatory mechanism of this auxiliary pathway. Such a regulation could serve to distribute the glycolytic flux over the two auxiliary branches as a response to the different environments that the parasite encounters during its life cycle.
Assuntos
Doença de Chagas/parasitologia , Piruvato Ortofosfato Diquinase/metabolismo , Trypanosoma cruzi/enzimologia , Monofosfato de Adenosina/metabolismo , Difosfatos/metabolismo , Glucose/metabolismo , Glicólise , Concentração de Íons de Hidrogênio , Cinética , Microcorpos/enzimologia , Fosfoenolpiruvato/metabolismo , Piruvato Ortofosfato Diquinase/química , Piruvatos/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
Per-Arnt-Sim (PAS) domains are structurally conserved and present in numerous proteins throughout all branches of the phylogenetic tree. Although PAS domain-containing proteins are major players for the adaptation to environmental stimuli in both prokaryotic and eukaryotic organisms, these types of proteins are still uncharacterized in the trypanosomatid parasites, Trypanosome and Leishmania In addition, PAS-containing phosphoglycerate kinase (PGK) protein is uncharacterized in the literature. Here, we report a PAS domain-containing PGK (LmPAS-PGK) in the unicellular pathogen Leishmania The modeled structure of N-terminal of this protein exhibits four antiparallel ß sheets centrally flanked by α helices, which is similar to the characteristic signature of PAS domain. Activity measurements suggest that acidic pH can directly stimulate PGK activity. Localization studies demonstrate that the protein is highly enriched in the glycosome and its presence can also be seen in the lysosome. Gene knockout, overexpression and complement studies suggest that LmPAS-PGK plays a fundamental role in cell survival through autophagy. Furthermore, the knockout cells display a marked decrease in virulence when host macrophage and BALB/c mice were infected with them. Our work begins to clarify how acidic pH-dependent ATP generation by PGK is likely to function in cellular adaptability of Leishmania.
Assuntos
Autofagossomos/imunologia , Leishmania major , Macrófagos , Modelos Moleculares , Fosfoglicerato Quinase , Proteínas de Protozoários , Animais , Leishmania major/genética , Leishmania major/imunologia , Leishmania major/patogenicidade , Macrófagos/imunologia , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Fosfoglicerato Quinase/química , Fosfoglicerato Quinase/deficiência , Fosfoglicerato Quinase/imunologia , Estrutura Secundária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologiaRESUMO
Expression of the intracellular form amastigote specific genes in the Leishmania donovani parasite plays a major role in parasite replication in the macrophage. In the current work, we have characterized a novel hypothetical gene, Ld30b that is specifically transcribed in the intracellular stage of the parasite. The recombinant Ld30b protein exists as a pentamer in solution as identified by native-PAGE and size exclusion gel chromatography. Structural analysis using circular dichroism and molecular modeling indicate that Ld30b belongs to family of cAMP-dependent protein kinase type I-alpha regulatory subunit. Co-localization immunofluorescence microscopy and western blot analyses (using anti-Ld30b antibody and anti-hypoxanthine-guanine phosphoribosyl transferase, a glycosome marker) on the isolated parasite glycosome organelle fractions show that Ld30b is localized in glycosome, though lacked a glycosome targeting PTS1/2 signal in the protein sequence. Episomal expression of Ld30b in the parasite caused the arrest of promastigotes and amastigotes growth in vitro. Cell cycle analysis using flow cytometry indicates that these parasites are arrested in 'sub G0/G1' phase of the cell cycle. Single allele knockout of Ld30b in the parasite similarly attenuated its growth by accumulation of cells in the S phase of cell cycle, thus confirming the probable importance of appropriate level of protein in the cells. Studying such intracellular stage expressing genes might unravel novel regulatory pathways for the development of drugs or vaccine candidates against leishmaniasis.
Assuntos
Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Leishmania donovani/fisiologia , Ciclo Celular , Dicroísmo Circular , Clonagem Molecular , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/química , Regulação da Expressão Gênica no Desenvolvimento , Leishmania donovani/genética , Microcorpos/química , Microcorpos/metabolismo , Modelos Moleculares , Filogenia , Multimerização Proteica , Estrutura Secundária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
The major mammalian bloodstream form of the African sleeping sickness parasite Trypanosoma brucei multiplies rapidly, and it is important to understand how these cells divide. Organelle inheritance involves complex spatiotemporal re-arrangements to ensure correct distribution to daughter cells. Here, serial block face scanning electron microscopy (SBF-SEM) was used to reconstruct whole individual cells at different stages of the cell cycle to give an unprecedented temporal, spatial and quantitative view of organelle division, inheritance and abscission in a eukaryotic cell. Extensive mitochondrial branching occurred only along the ventral surface of the parasite, but the mitochondria returned to a tubular form during cytokinesis. Fission of the mitochondrion occurred within the cytoplasmic bridge during the final stage of cell division, correlating with cell abscission. The nuclei were located underneath each flagellum at mitosis and the mitotic spindle was located along the ventral surface, further demonstrating the asymmetric arrangement of cell cleavage in trypanosomes. Finally, measurements demonstrated that multiple Golgi bodies were accurately positioned along the flagellum attachment zone, suggesting a mechanism for determining the location of Golgi bodies along each flagellum during the cell cycle.
Assuntos
Ciclo Celular , Imageamento Tridimensional , Microscopia Eletrônica de Varredura/métodos , Organelas/metabolismo , Organelas/ultraestrutura , Trypanosoma brucei brucei/citologia , Trypanosoma brucei brucei/ultraestrutura , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Flagelos/metabolismo , Flagelos/ultraestrutura , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial , Modelos Biológicos , Trypanosoma brucei brucei/metabolismoRESUMO
Protein import into the Leishmania glycosome requires docking of the cargo-loaded peroxin 5 (PEX5) receptor to the peroxin 14 (PEX14) bound to the glycosome surface. To examine the LdPEX14-membrane interaction, we purified L. donovani promastigote glycosomes and determined the phospholipid and fatty acid composition. These membranes contained predominately phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol (PG) modified primarily with C18 and C22 unsaturated fatty acid. Using large unilamellar vesicles (LUVs) with a lipid composition mimicking the glycosomal membrane in combination with sucrose density centrifugation and fluorescence-activated cell sorting technique, we established that the LdPEX14 membrane-binding activity was dependent on a predicted transmembrane helix found within residues 149-179. Monolayer experiments showed that the incorporation of PG and phospholipids with unsaturated fatty acids, which increase membrane fluidity and favor a liquid expanded phase, facilitated the penetration of LdPEX14 into biological membranes. Moreover, we demonstrated that the binding of LdPEX5 receptor or LdPEX5-PTS1 receptor-cargo complex was contingent on the presence of LdPEX14 at the surface of LUVs.
Assuntos
Leishmania donovani/metabolismo , Microcorpos/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos/química , Fosfatidilgliceróis/química , Proteínas de Protozoários/química , Sequência de Aminoácidos , Sítios de Ligação , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Fracionamento Celular , Colesterol/química , Colesterol/metabolismo , Expressão Gênica , Interações Hidrofóbicas e Hidrofílicas , Leishmania donovani/genética , Fluidez de Membrana , Microcorpos/química , Receptor 1 de Sinal de Orientação para Peroxissomos/genética , Receptor 1 de Sinal de Orientação para Peroxissomos/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Fosfatidilgliceróis/metabolismo , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismoRESUMO
To facilitate a greater understanding of the biological processes in the medically important Leishmania donovani parasite, a combination of differential and density-gradient ultracentrifugation techniques were used to achieve a comprehensive subcellular fractionation of the promastigote stage. An in-depth label-free proteomic LC-MS/MS analysis of the density gradients resulted in the identification of â¼50% of the Leishmania proteome (3883 proteins detected), which included â¼645 integral membrane proteins and 1737 uncharacterized proteins. Clustering and subcellular localization of proteins was based on a subset of training Leishmania proteins with known subcellular localizations that had been determined using biochemical, confocal microscopy, or immunoelectron microscopy approaches. This subcellular map will be a valuable resource that will help dissect the cell biology and metabolic processes associated with specific organelles of Leishmania and related kinetoplastids.
Assuntos
Leishmania donovani/química , Proteínas de Membrana/isolamento & purificação , Redes e Vias Metabólicas/genética , Proteoma/isolamento & purificação , Proteômica/métodos , Proteínas de Protozoários/isolamento & purificação , Fracionamento Celular/instrumentação , Fracionamento Celular/métodos , Núcleo Celular/química , Núcleo Celular/metabolismo , Centrifugação com Gradiente de Concentração , Cromatografia Líquida , Expressão Gênica , Ontologia Genética , Leishmania donovani/genética , Leishmania donovani/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microcorpos/química , Microcorpos/metabolismo , Microssomos/química , Microssomos/metabolismo , Mitocôndrias/química , Mitocôndrias/metabolismo , Anotação de Sequência Molecular , Proteoma/genética , Proteoma/metabolismo , Proteômica/instrumentação , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Frações Subcelulares , Espectrometria de Massas em Tandem , UltracentrifugaçãoRESUMO
Glycerophospholipids are the main constituents of the biological membranes in Trypanosoma brucei, which causes sleeping sickness in humans. The present work reports the characterization of the alkyl-dihydroxyacetonephosphate synthase TbADS that catalyzes the committed step in ether glycerophospholipid biosynthesis. TbADS localizes to the glycosomal lumen. TbADS complemented a null mutant of Leishmania major lacking alkyl-dihydroxyacetonephosphate synthase activity and restored the formation of normal form of the ether lipid based virulence factor lipophosphoglycan. Despite lacking alkyl-dihydroxyacetonephosphate synthase activity, a null mutant of TbADS in procyclic trypanosomes remained viable and exhibited normal growth. Comprehensive analysis of cellular glycerophospholipids showed that TbADS was involved in the biosynthesis of all ether glycerophospholipid species, primarily found in the PE and PC classes.
Assuntos
Alquil e Aril Transferases/metabolismo , Glicerofosfolipídeos/biossíntese , Leishmania major/enzimologia , Microcorpos/enzimologia , Trypanosoma brucei brucei/enzimologia , Leishmania major/genética , Leishmania major/metabolismo , Mutação com Perda de Função , Plasmídeos/química , Plasmídeos/genética , Plasmídeos/metabolismo , Espectrometria de Massas em Tandem , Trypanosoma brucei brucei/metabolismoRESUMO
Isocitrate dehydrogenases (IDHs) are metabolic enzymes that catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate. Depending on the electron acceptor and subcellular localization, these enzymes are classified as NADP+-dependent IDH1 in the cytosol or peroxisomes, NADP+-dependent IDH2 and NAD+-dependent IDH3 in mitochondria. Trypanosoma brucei is a protozoan parasite that causes African sleeping sickness in humans and Nagana disease in animals. Here, for the first time, a putative glycosomal T. brucei type 1 IDH (TbIDH1) was expressed in Escherichia coli and purified for crystallographic study. Surprisingly, the putative NADP+-dependent TbIDH1 has higher activity with NAD+ compared with NADP+ as electron acceptor, a unique characteristic among known eukaryotic IDHs which encouraged us to crystallize TbIDH1 for future biochemical and structural studies. Methods of expression and purification of large amounts of recombinant TbIDH1 with improved solubility to facilitate protein crystallization are presented.
Assuntos
Isocitrato Desidrogenase/genética , NADP/metabolismo , NAD/metabolismo , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/genética , Trypanosoma brucei brucei/química , Sequência de Aminoácidos , Clonagem Molecular , Cristalização , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Isocitrato Desidrogenase/isolamento & purificação , Isocitrato Desidrogenase/metabolismo , Isocitratos/metabolismo , Ácidos Cetoglutáricos/metabolismo , Peso Molecular , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Trypanosoma brucei brucei/enzimologiaRESUMO
Acetylation is a ubiquitous protein modification present in prokaryotic and eukaryotic cells that participates in the regulation of many cellular processes. The bromodomain is the only domain known to bind acetylated lysine residues. In the last few years, many bromodomain inhibitors have been developed in order to treat diseases caused by aberrant acetylation of lysine residues and have been tested as anti-parasitic drugs. In the present paper, we report the first characterization of Trypanosoma cruzi bromodomain factor 1 (TcBDF1). TcBDF1 is expressed in all life cycle stages, but it is developmentally regulated. It localizes in the glycosomes directed by a PTS2 (peroxisome-targeting signal 2) sequence. The overexpression of wild-type TcBDF1 is detrimental for epimastigotes, but it enhances the infectivity rate of trypomastigotes and the replication of amastigotes. On the other hand, the overexpression of a mutated version of TcBDF1 has no effect on epimastigotes, but it does negatively affect trypomastigotes' infection and amastigotes' replication.
Assuntos
Líquido Intracelular/metabolismo , Proteínas de Membrana/biossíntese , Microcorpos/metabolismo , Neuraminidase/biossíntese , Proteínas de Protozoários/biossíntese , Trypanosoma cruzi/metabolismo , Animais , Chlorocebus aethiops , Líquido Intracelular/parasitologia , Microcorpos/parasitologia , Células VeroRESUMO
Protozoan parasites of the family Trypanosomatidae infect humans as well as livestock causing devastating diseases like sleeping sickness, Chagas disease, and Leishmaniasis. These parasites compartmentalize glycolytic enzymes within unique organelles, the glycosomes. Glycosomes represent a subclass of peroxisomes and they are essential for the parasite survival. Hence, disruption of glycosome biogenesis is an attractive drug target for these Neglected Tropical Diseases (NTDs). Peroxin 16 (PEX16) plays an essential role in peroxisomal membrane protein targeting and de novo biogenesis of peroxisomes from endoplasmic reticulum (ER). We identified trypanosomal PEX16 based on specific sequence characteristics and demonstrate that it is an integral glycosomal membrane protein of procyclic and bloodstream form trypanosomes. RNAi mediated partial knockdown of Trypanosoma brucei PEX16 in bloodstream form trypanosomes led to severe ATP depletion, motility defects and cell death. Microscopic and biochemical analysis revealed drastic reduction in glycosome number and mislocalization of the glycosomal matrix enzymes to the cytosol. Asymmetry of the localization of the remaining glycosomes was observed with a severe depletion in the posterior part. The results demonstrate that trypanosomal PEX16 is essential for glycosome biogenesis and thereby, provides a potential drug target for sleeping sickness and related diseases.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Peroxissomos/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Animais , Retículo Endoplasmático/genética , Humanos , Proteínas de Membrana/genética , Peroxissomos/genética , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/genética , Tripanossomíase Africana/tratamento farmacológico , Tripanossomíase Africana/genética , Tripanossomíase Africana/metabolismo , Tripanossomíase Africana/patologiaRESUMO
BACKGROUND: The glycosome is a unique organelle found in Kinetoplastids known to compartmentalize vital metabolic pathways including glycolysis, ß-fatty acid oxidation and purine salvage. Organelle biogenesis depends on a network of proteins for trafficking and translocation of nascent protein into the glycosome. The interaction of the proteins LdPEX14 and LdPEX5 at the glycosome membrane is crucial for targeting proteins into this organelle. METHODS: Deletion mutagenesis, pull-down, and bacterial two hybrid assay were used to map the LdPEX5 domain bound by LdPEX14. ELISA assays, ITC, intrinsic fluorescence and size exclusion chromatography to monitor binding and structural changes associated with the LdPEX5-LdPEX14 interaction. RESULTS AND CONCLUSIONS: The LdPEX14 binding site was mapped to residues 280-300 on LdPEX5, a region containing the pentapeptide motif W(293)AQEY(297). Deletion of this region abolished the LdPEX5-LdPEX14 interaction. Intrinsic fluorescence spectroscopy suggests that the stabilization of the LdPEX5-LdPEX14 complex is dependent on W293 docking into a hydrophobic pocket within the binding domain of ldpex14. Studies using a panel of synthetic peptides suggest a critical role for Y297 and to a lesser extent E296 in stabilizing the LdPEX5-LdPEX14 association. GENERAL SIGNIFICANCE: We show that the LdPEX14 binding site is more promiscuous and in contrast to other eukaryotic systems will accommodate a more degenerate pentapeptide motif with the sequences WXXXW or FXXXF, findings which may be exploited for potential drug design.
Assuntos
Fragmentos de Peptídeos/química , Proteínas de Protozoários/química , Receptores Citoplasmáticos e Nucleares/química , Motivos de Aminoácidos , Sítios de Ligação , Receptor 1 de Sinal de Orientação para Peroxissomos , Estabilidade Proteica , Estrutura Quaternária de ProteínaRESUMO
Trypanosoma cruzi, like other trypanosomatids analyzed so far, can use both glucose and amino acids as carbon and energy source. In these parasites, glycolysis is compartmentalized in glycosomes, authentic but specialized peroxisomes. The major part of this pathway, as well as a two-branched glycolytic auxiliary system, are present in these organelles. The first enzyme of one branch of this auxiliary system is the PPi-dependent pyruvate phosphate dikinase (PPDK) that converts phosphoenolpyruvate (PEP), inorganic pyrophosphate (PPi) and AMP into pyruvate, inorganic phosphate (Pi) and ATP, thus contributing to the ATP/ADP balance within the glycosomes. In this work we cloned, expressed and purified the T. cruzi PPDK. It kinetic parameters were determined, finding KM values for PEP, PPi and AMP of 320, 70 and 17 µM, respectively. Using molecular exclusion chromatography, two native forms of the enzyme were found with estimated molecular weights of 200 and 100 kDa, corresponding to a homodimer and monomer, respectively. It was established that T. cruzi PPDK's specific activity can be enhanced up to 2.6 times by the presence of ammonium in the assay mixture. During growth of epimastigotes in batch culture an apparent decrease in the specific activity of PPDK was observed. However, when its activity is normalized for the presence of ammonium in the medium, no significant modification of the enzyme activity per cell in time was found.
Assuntos
Piruvato Ortofosfato Diquinase/metabolismo , Trypanosoma cruzi/enzimologia , Cloreto de Amônio/metabolismo , Animais , Doença de Chagas/parasitologia , Clonagem Molecular , Escherichia coli , Regulação Enzimológica da Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Cinética , Magnésio/metabolismo , Microcorpos/metabolismo , Peso Molecular , Cloreto de Potássio/metabolismo , Piruvato Ortofosfato Diquinase/química , Piruvato Ortofosfato Diquinase/genética , Piruvato Ortofosfato Diquinase/isolamento & purificação , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Cloreto de Sódio/metabolismo , Trypanosoma cruzi/genéticaRESUMO
Trypanosoma evansi is a monomorphic protist that can infect horses and other animal species of economic importance for man. Like the bloodstream form of the closely related species Trypanosoma brucei, T. evansi depends exclusively on glycolysis for its free-energy generation. In T. evansi as in other kinetoplastid organisms, the enzymes of the major part of the glycolytic pathway are present within organelles called glycosomes, which are authentic but specialized peroxisomes. Since T. evansi does not undergo stage-dependent differentiations, it occurs only as bloodstream forms, it has been assumed that the metabolic pattern of this parasite is identical to that of the bloodstream form of T. brucei. However, we report here the presence of two additional enzymes, phosphoenolpyruvate carboxykinase and PPi-dependent pyruvate phosphate dikinase in T. evansi glycosomes. Their colocalization with glycolytic enzymes within the glycosomes of this parasite has not been reported before. Both enzymes can make use of PEP for contributing to the production of ATP within the organelles. The activity of these enzymes in T. evansi glycosomes drastically changes the model assumed for the oxidation of glucose by this parasite.
Assuntos
Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Piruvato Ortofosfato Diquinase/metabolismo , Trypanosoma/enzimologia , Animais , Digitonina/farmacologia , Glucosefosfato Desidrogenase/isolamento & purificação , Glucosefosfato Desidrogenase/metabolismo , Glicólise , Hexoquinase/isolamento & purificação , Hexoquinase/metabolismo , Cavalos , Indicadores e Reagentes/farmacologia , Malato Desidrogenase/isolamento & purificação , Malato Desidrogenase/metabolismo , Camundongos , Microcorpos/enzimologia , Microscopia de Fluorescência , Permeabilidade/efeitos dos fármacos , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/isolamento & purificação , Fosfoglicerato Quinase/isolamento & purificação , Fosfoglicerato Quinase/metabolismo , Fosfopiruvato Hidratase/isolamento & purificação , Fosfopiruvato Hidratase/metabolismo , Piruvato Ortofosfato Diquinase/isolamento & purificação , Coelhos , Ratos , Ratos Wistar , Trypanosoma/efeitos dos fármacosRESUMO
Gluconeogenesis is an active pathway in Leishmania amastigotes and is essential for their survival within the mammalian cells. However, our knowledge about this pathway in trypanosomatids is very limited. We investigated the role of glycerol kinase (GK), phosphoenolpyruvate carboxykinase (PEPCK), and pyruvate phosphate dikinase (PPDK) in gluconeogenesis by generating the respective Leishmania mexicana Δgk, Δpepck, and Δppdk null mutants. Our results demonstrated that indeed GK, PEPCK, and PPDK are key players in the gluconeogenesis pathway in Leishmania, although stage-specific differences in their contribution to this pathway were found. GK participates in the entry of glycerol in promastigotes and amastigotes; PEPCK participates in the entry of aspartate in promastigotes, and PPDK is involved in the entry of alanine in amastigotes. Furthermore, the majority of alanine enters into the pathway via decarboxylation of pyruvate in promastigotes, whereas pathway redundancy is suggested for the entry of aspartate in amastigotes. Interestingly, we also found that l-lactate, an abundant glucogenic precursor in mammals, was used by Leishmania amastigotes to synthesize mannogen, entering the pathway through PPDK. On the basis of these new results, we propose a revision in the current model of gluconeogenesis in Leishmania, emphasizing the differences between amastigotes and promastigotes. This work underlines the importance of studying the trypanosomatid intracellular life cycle stages to gain a better understanding of the pathologies caused in humans.
Assuntos
Gluconeogênese , Glicerol Quinase/metabolismo , Leishmania mexicana/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Proteínas de Protozoários/metabolismo , Piruvato Ortofosfato Diquinase/metabolismo , Vias Biossintéticas/efeitos dos fármacos , Southern Blotting , Western Blotting , DNA de Protozoário/genética , Glucose/metabolismo , Glucose/farmacologia , Glicerol Quinase/genética , Humanos , Leishmania mexicana/genética , Leishmania mexicana/crescimento & desenvolvimento , Estágios do Ciclo de Vida , Mutação , Fosfoenolpiruvato Carboxilase/genética , Proteínas de Protozoários/genética , Piruvato Ortofosfato Diquinase/genéticaRESUMO
Leishmania contains two phosphoglycerate kinase (PGK) genes, PGKB and PGKC, which code for the cytosolic and glycosomal isoforms of the enzyme, respectively. Although differences in PGKB and PGKC transcript and protein levels and isoform activities have been well documented, the mechanisms of control of both transcript and protein abundance have not been described to date. To better understand the regulation of Leishmania PGK expression, we investigated the stabilities of both PGK transcripts using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) in combination with transcription and trans-splicing inhibitors. Cells were treated with sinefungin and actinomycin D, and RNA decay kinetics were assessed. In addition, immunoblotting and protein synthesis inhibition by cycloheximide were employed to evaluate protein steady states and degradation. We observed increased stabilities of both PGKB mRNA and protein compared with the glycosomal isoform (PGKC). Our results indicate that both post-transcriptional and post-translational events contribute to the distinct expression levels of the PGKB and PGKC isoforms in Leishmania major.
Assuntos
Leishmania major/enzimologia , Fosfoglicerato Quinase/genética , Adenosina/análogos & derivados , Adenosina/farmacologia , Antiprotozoários/farmacologia , Cicloeximida/farmacologia , Citosol/enzimologia , Dactinomicina/farmacologia , Regulação da Expressão Gênica , Meia-Vida , Immunoblotting , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Leishmania major/efeitos dos fármacos , Leishmania major/genética , Microcorpos/enzimologia , Peso Molecular , Fosfoglicerato Quinase/química , Fosfoglicerato Quinase/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , RNA de Protozoário/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
Trypanosomatids contain peroxisome-like organelles called glycosomes. Peroxisomal biogenesis involves a cytosolic receptor, PEX5, which, after its insertion into the organellar membrane, delivers proteins to the matrix. In yeasts and mammalian cells, transient PEX5 monoubiquitination at the membrane serves as the signal for its retrieval from the organelle for re-use. When its recycling is impaired, PEX5 is polyubiquitinated for proteasomal degradation. Stably monoubiquitinated TbPEX5 was detected in cytosolic fractions of Trypanosoma brucei, indicative for its role as physiological intermediate in receptor recycling. This modification's resistance to dithiothreitol suggests ubiquitin conjugation of a lysine residue. T. brucei PEX4, the functional homologue of the ubiquitin-conjugating (UBC) enzyme responsible for PEX5 monoubiquitination in yeast, was identified. It is associated with the cytosolic face of the glycosomal membrane, probably anchored by an identified putative TbPEX22. The involvement of TbPEX4 in TbPEX5 ubiquitination was demonstrated using procyclic ∆PEX4 trypanosomes. Surprisingly, glycosomal matrix protein import was only mildly affected in this mutant. Since other UBC homologues were upregulated, it might be possible that these have partially rescued PEX4's function in PEX5 ubiquitination. In addition, the altered expression of UBCs, notably of candidates involved in cell-cycle control, could be responsible for observed morphological and motility defects of the ∆PEX4 mutant.
Assuntos
Microcorpos/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Ubiquitinação , Animais , Linhagem Celular , Citosol/metabolismo , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Membranas Intracelulares/metabolismo , Estágios do Ciclo de Vida , Modelos Biológicos , Transporte Proteico , Proteínas de Protozoários/genética , Reprodutibilidade dos Testes , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/ultraestrutura , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Trypanosomatid parasites are kinetoplastid protists that compartmentalize glycolytic enzymes in unique peroxisome-related organelles called glycosomes. The heterohexameric AAA-ATPase complex of PEX1-PEX6 is anchored to the peroxisomal membrane and functions in the export of matrix protein import receptor PEX5 from the peroxisomal membrane. Defects in PEX1, PEX6 or their membrane anchor causes dysfunction of peroxisomal matrix protein import cycle. In this study, we functionally characterized a putative Trypanosoma PEX1 orthologue by bioinformatic and experimental approaches and show that it is a true PEX1 orthologue. Using yeast two-hybrid analysis, we demonstrate that TbPEX1 can bind to TbPEX6. Endogenously tagged TbPEX1 localizes to glycosomes in the T. brucei parasites. Depletion of PEX1 gene expression by RNA interference causes lethality to the bloodstream form trypanosomes, due to a partial mislocalization of glycosomal enzymes to the cytosol and ATP depletion. TbPEX1 RNAi leads to a selective proteasomal degradation of both matrix protein import receptors TbPEX5 and TbPEX7. Unlike in yeast, PEX1 depletion did not result in an accumulation of ubiquitinated TbPEX5 in trypanosomes. As PEX1 turned out to be essential for trypanosomatid parasites, it could provide a suitable drug target for parasitic diseases. The results also suggest that these parasites possess a highly efficient quality control mechanism that exports the import receptors from glycosomes to the cytosol in the absence of a functional TbPEX1-TbPEX6 complex.
Assuntos
Parasitos , Proteínas de Saccharomyces cerevisiae , Trypanosoma , Animais , Parasitos/metabolismo , Saccharomyces cerevisiae/metabolismo , Peroxissomos/genética , Peroxissomos/metabolismo , Microcorpos , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Kinetoplastids, a group of flagellated protists that are often insect intestinal parasites, encounter various sources of oxidative stress. Such stressors include reactive oxygen species, both internally produced within the protist, and induced externally by host immune responses. This investigation focuses on the role of a highly conserved aspartate-based protein phosphatase, PTP-Interacting protein (PIP39) in managing oxidative stress. In addition to its well accepted role in a Trypanosoma brucei life stage transition, there is evidence of PIP39 participation in the T. brucei oxidative stress response. To examine whether this latter PIP39 role may exist more broadly, we aimed to elucidate PIP39's contribution to redox homeostasis in the monoxenous parasite Leptomonas seymouri. Utilizing CRISPR-Cas9-mediated elimination of PIP39 in conjunction with oxidative stress assays, we demonstrate that PIP39 is required for cellular tolerance to oxidative stress in L. seymouri, positing it as a putative regulatory node for adaptive stress responses. We propose that future analysis of L. seymouri PIP39 enzymatic activity, regulation, and potential localization to a specialized organelle termed a glycosome will contribute to a deeper understanding of the molecular mechanisms by which protozoan parasites adapt to oxidative environments. Our study also demonstrates success at using gene editing tools developed for Leishmania for the related L. seymouri.
Assuntos
Estresse Oxidativo , Proteínas de Protozoários , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Sistemas CRISPR-Cas , Kinetoplastida/genética , Kinetoplastida/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas Fosfatases/genética , Espécies Reativas de Oxigênio/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/fisiologiaRESUMO
PEX19 binding sites are essential parts of the targeting signals of peroxisomal membrane proteins (mPTS). In this study, we characterized PEX19 binding sites of PEX11, the most abundant peroxisomal and glycosomal membrane protein from Trypanosoma brucei and Saccharomyces cerevisiae. TbPEX11 contains two PEX19 binding sites, one close to the N-terminus (BS1) and a second in proximity to the first transmembrane domain (BS2). The N-terminal BS1 is highly conserved across different organisms and is required for maintenance of the steady-state concentration and efficient targeting to peroxisomes and glycosomes in both baker's yeast and Trypanosoma brucei. The second PEX19 binding site in TbPEX11 is essential for its glycosomal localization. Deletion or mutations of the PEX19 binding sites in TbPEX11 or ScPEX11 results in mislocalization of the proteins to mitochondria. Bioinformatic analysis indicates that the N-terminal region of TbPEX11 contains an amphiphilic helix and several putative TOM20 recognition motifs. We show that the extreme N-terminal region of TbPEX11 contains a cryptic N-terminal signal that directs PEX11 to the mitochondrion if its glycosomal transport is blocked.