RESUMO
Toxoplasma gondii is an apicomplexan parasite that infects and proliferates within many different types of host cells and infects virtually all warm-blooded animals and humans. Trypanosoma brucei is an extracellular kinetoplastid that causes human African trypanosomiasis and Nagana disease in cattle, primarily in rural sub-Saharan Africa. Current treatments against both parasites have limitations, e.g., suboptimal efficacy and adverse side effects. Here, we investigate the potential cellular and molecular targets of a trithiolato-bridged arene ruthenium complex conjugated to 9-(2-hydroxyethyl)-adenine (1), which inhibits both parasites with IC50s below 10-7 M. Proteins that bind to 1 were identified using differential affinity chromatography (DAC) followed by shotgun-mass spectrometry. A trithiolato-bridged ruthenium complex decorated with hypoxanthine (2) and 2-hydroxyethyl-adenine (3) were included as controls. Transmission electron microscopy (TEM) revealed distinct ultrastructural modifications in the mitochondrion induced by (1) but not by (2) and (3) in both species. DAC revealed 128 proteins in T. gondii and 46 proteins in T. brucei specifically binding to 1 but not 2 or 3. In T. gondii, the most abundant was a protein with unknown function annotated as YOU2. This protein is a homolog to the human mitochondrial inner membrane translocase subunit Tim10. In T. brucei, the most abundant proteins binding specifically to 1 were mitochondrial ATP-synthase subunits. Exposure of T. brucei bloodstream forms to 1 resulted in rapid breakdown of the ATP-synthase complex. Moreover, both datasets contained proteins involved in key steps of metabolism and nucleic acid binding proteins.
Assuntos
Nucleotídeos/química , Compostos de Rutênio/farmacologia , Compostos de Sulfidrila/química , Toxoplasma/efeitos dos fármacos , Toxoplasmose/tratamento farmacológico , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase/tratamento farmacológico , Humanos , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Proteínas de Protozoários/metabolismo , Compostos de Rutênio/química , Toxoplasma/metabolismo , Toxoplasmose/metabolismo , Toxoplasmose/parasitologia , Trypanosoma brucei brucei/metabolismo , Tripanossomíase/metabolismo , Tripanossomíase/parasitologiaRESUMO
Leucinostatin A is one of the most potent antiprotozoal compounds ever described, but little was known on structure-activity relationships (SAR). We used Trypanosoma brucei as a protozoal model organism to test synthetically modified derivatives, resulting in simplified but equally active compounds 2 (ZHAWOC6025) and 4 (ZHAWOC6027), which were subsequently modified in all regions of the molecule to gain an in-depth SAR understanding. The antiprotozoal SAR matched SAR in phospholipid liposomes, where membrane integrity, leaking, and dynamics were studied. The mode of action is discussed based on a structure-activity analysis of derivatives in efficacy, ultrastructural studies in T. brucei, and artificial membrane models, mimicking membrane stability and membrane potential. The main site of antiprotozoal action of natural and synthetic leucinostatins lies in the destabilization of the inner mitochondrial membrane, as demonstrated by ultrastructural analysis, electron microscopy and mitochondrial staining. Long-time sublethal exposure of T. brucei (200 passages) and siRNA screening of 12'000 mutants showed no signs of resistance development to the synthetic derivatives.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Antiprotozoários/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/química , Antiprotozoários/síntese química , Antiprotozoários/química , Conformação Molecular , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade , Trypanosoma brucei brucei/genéticaRESUMO
Mammalian phosphatidic acid phosphatases, also called lipins, show high amino acid sequence identity to Saccharomyces cerevisiae Pah1p and catalyze the dephosphorylation of phosphatidic acid (PA) to diacylglycerol. Both the substrate and product of the reaction are key precursors for the synthesis of phospholipids and triacylglycerol (TAG). We now show that expression of the Trypanosoma brucei lipin homolog TbLpn is essential for parasite survival in culture. Inducible down-regulation of TbLpn in T. brucei procyclic forms increased cellular PA content, decreased the numbers of lipid droplets, reduced TAG steady-state levels and inhibited in vivo [3 H]TAG formation after labeling trypanosomes with [3 H]glycerol. In addition, fluorescence and transmission electron microscopy revealed that depletion of TbLpn caused major alterations in mitochondrial morphology and function, i.e., the appearance of distorted mitochondrial matrix, and reduced ATP production via oxidative phosphorylation. Effects of lipin depletion on mitochondrial integrity have previously not been reported. N- and C-terminally tagged forms of TbLpn were localized in the cytosol.
RESUMO
Trypanosoma brucei causes human African trypanosomiasis and Nagana disease in cattle, imposing substantial medical and economic burden in sub-Saharan Africa. The current treatments have limitations, including the requirement for elaborated protocols, development of drug resistance, and they are prone to adverse side effects. In vitro screening of a library of 14 dinuclear-thiolato bridged arene ruthenium complexes, originally developed for treatment of cancer cells, resulted in the identification of 7 compounds with IC50 values ranging from 3 to 26â¯nM. Complex [(η6-p-MeC6H4Pri)2Ru2(µ2-SC6H4-o-Pri)3]Cl (2) (IC50â¯=â¯4â¯nM) and complex [(η6-p-MeC6H4Pri)2Ru2(µ2-SCH2C6H4-p-But)2(µ2-SC6H4-p-OH)]BF4(9) (IC50â¯=â¯26â¯nM) were chosen for further assessments. Application of complex 2 and 9â¯at 20â¯nM and 200â¯nM, respectively, for 4.5â¯h induced alterations in the trypanosome mitochondrion as evidenced by immunofluorescence employing an antibody against mitochondrial Hsp70 and Mitotracker labeling. Transmission electron microscopy of parasites taken at 2 and 4h of treatment demonstrated massive alterations in the mitochondrial ultrastructure, while other organelles and structural elements of the parasites remained unaffected. Complex 2 treated trypanosomes exhibited a distorted mitochondrial membrane, and the mitochondrial matrix was transformed into an amorphous mass with different degrees of electron densities. Complex 9 did not notably impair the integrity of the membrane, but the interior of the mitochondrion appeared either completely translucent, or was filled with filamentous structures of unknown nature. Dose- and time-dependent effects of these two compounds on the mitochondrial membrane potential were detected by tetramethylrhodamine ethyl ester assay. Thus, the mitochondrion and associated metabolic processes are an important target of dinuclear thiolato-bridged arene ruthenium complexes in T. brucei.
Assuntos
Potencial da Membrana Mitocondrial/efeitos dos fármacos , Compostos de Rutênio/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase Africana/parasitologia , Animais , Relação Dose-Resposta a Droga , Imunofluorescência , Humanos , Concentração Inibidora 50 , Microscopia Eletrônica de Transmissão , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Compostos de Rutênio/química , Fatores de Tempo , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/ultraestrutura , Tripanossomíase Africana/sangueRESUMO
Phosphatidylethanolamine (PE) and phosphatidylserine (PS) are ubiquitously expressed and metabolically interconnected glycerophospholipids in eukaryotes and prokaryotes. In Trypanosoma brucei, PE synthesis has been shown to occur mainly via the Kennedy pathway, one of the three routes leading to PE synthesis in eukaryotes, while PS synthesis has not been studied experimentally. We now reveal the importance of T. brucei PS synthase 2 (TbPSS2) and T. brucei PS decarboxylase (TbPSD), two key enzymes involved in aminophospholipid synthesis, for trypanosome viability. By using tetracycline-inducible down-regulation of gene expression and in vivo and in vitro metabolic labeling, we found that TbPSS2 (i) is necessary for normal growth of procyclic trypanosomes, (ii) localizes to the endoplasmic reticulum and (iii) represents the unique route for PS formation in T. brucei. In addition, we identified TbPSD as type I PS decarboxylase in the mitochondrion and found that it is processed proteolytically at a WGSS cleavage site into a heterodimer. Down-regulation of TbPSD expression affected mitochondrial integrity in both procyclic and bloodstream form trypanosomes, decreased ATP production via oxidative phosphorylation in procyclic form and affected parasite growth.
Assuntos
Carboxiliases/metabolismo , Transferases de Grupos Nitrogenados/metabolismo , Trypanosoma brucei brucei/enzimologia , Descarboxilação , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/genéticaRESUMO
Mature dolichol-linked oligosaccharides (mDLOs) needed for eukaryotic protein N-glycosylation are synthesized by a multistep pathway in which the biosynthetic lipid intermediate Man5GlcNAc2-PP-dolichol (M5-DLO) flips from the cytoplasmic to the luminal face of the endoplasmic reticulum. The endoplasmic reticulum membrane protein Rft1 is intimately involved in mDLO biosynthesis. Yeast genetic analyses implicated Rft1 as the M5-DLO flippase, but because biochemical tests challenged this assignment, the function of Rft1 remains obscure. To understand the role of Rft1, we sought to analyze mDLO biosynthesis in vivo in the complete absence of the protein. Rft1 is essential for yeast viability, and no Rft1-null organisms are currently available. Here, we exploited Trypanosoma brucei (Tb), an early diverging eukaryote whose Rft1 homologue functions in yeast. We report that TbRft1-null procyclic trypanosomes grow nearly normally. They have normal steady-state levels of mDLO and significant N-glycosylation, indicating robust M5-DLO flippase activity. Remarkably, the mutant cells have 30-100-fold greater steady-state levels of M5-DLO than wild-type cells. All N-glycans in the TbRft1-null cells originate from mDLO indicating that the M5-DLO excess is not available for glycosylation. These results suggest that rather than facilitating M5-DLO flipping, Rft1 facilitates conversion of M5-DLO to mDLO by another mechanism, possibly by acting as an M5-DLO chaperone.
Assuntos
Células Eucarióticas/metabolismo , Glicoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Eletroforese em Gel de Poliacrilamida , Retículo Endoplasmático/metabolismo , Citometria de Fluxo , Glucose/farmacologia , Glicoproteínas/genética , Glicosilação , Proteínas de Membrana Lisossomal/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Microscopia de Fluorescência , Modelos Biológicos , Mutação , Oligossacarídeos de Poli-Isoprenil Fosfato/metabolismo , Polissacarídeos/metabolismo , Biossíntese de Proteínas , Proteínas de Protozoários/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transformação Genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crescimento & desenvolvimentoRESUMO
Procyclins are abundant, glycosylphosphatidylinositol (GPI)-anchored proteins on the surface of procyclic (insect) form trypanosomes. To investigate whether trypanosomes are able to survive without a procyclin coat, all four procyclin genes were deleted sequentially. Bloodstream forms of the null mutant exhibited no detectable phenotype and were able to differentiate to procyclic forms. Initially, differentiated null mutant cells were barely able to grow, but after an adaptation period of 2 mo in culture they proliferated at the same rate as wild-type trypanosomes. Analysis of these culture-adapted null mutants revealed that they were covered by free GPIs. These were closely related to the mature procyclin anchor in structure and were expressed on the surface in numbers comparable with that of procyclin in wild-type cells. However, free GPIs were smaller than the procyclin anchor, indicative of a lower number of poly-N-acetyllactosamine repeats, and a proportion contained diacylphosphatidic acid. Free GPIs are also expressed by wild-type cells, although to a lesser extent. These have been overlooked in the past because they partition in a solvent fraction (chloroform/water/methanol) that is normally discarded when GPI-anchored proteins are purified.
Assuntos
Glicosilfosfatidilinositóis/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Animais , Membrana Celular/metabolismo , Deleção de Genes , Genes de Protozoários , Glicosilfosfatidilinositóis/química , Trypanosoma brucei brucei/crescimento & desenvolvimentoAssuntos
Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/fisiologia , Moscas Tsé-Tsé/parasitologia , Animais , Sequência de Bases , Primers do DNA , Deleção de Genes , Dados de Sequência Molecular , Fosforilação , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/isolamento & purificaçãoRESUMO
In addition to its conventional role during protein synthesis, eukaryotic elongation factor 1A is involved in other cellular processes. Several regions of interaction between eukaryotic elongation factor 1A and the translational apparatus or the cytoskeleton have been identified, yet the roles of the different post-translational modifications of eukaryotic elongation factor 1A are completely unknown. One amino acid modification, which so far has only been found in eukaryotic elongation factor 1A, consists of ethanolamine-phosphoglycerol attached to two glutamate residues that are conserved between mammals and plants. We now report that ethanolamine-phosphoglycerol is also present in eukaryotic elongation factor 1A of the protozoan parasite Trypanosoma brucei, indicating that this unique protein modification is of ancient origin. In addition, using RNA-mediated gene silencing against enzymes of the Kennedy pathway, we demonstrate that phosphatidylethanolamine is a direct precursor of the ethanolamine-phosphoglycerol moiety. Down-regulation of the expression of ethanolamine kinase and ethanolamine-phosphate cytidylyltransferase results in inhibition of phosphatidylethanolamine synthesis in T. brucei procyclic forms and, concomitantly, in a block in glycosylphosphatidylinositol attachment to procyclins and ethanolamine-phosphoglycerol modification of eukaryotic elongation factor 1A.
Assuntos
Etanolamina/metabolismo , Glicerofosfatos/metabolismo , Fator 1 de Elongação de Peptídeos/metabolismo , Fosfatidiletanolaminas/metabolismo , Sequência de Aminoácidos , Animais , Regulação para Baixo , Etanolamina/química , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Glicerofosfatos/química , Espectrometria de Massas , Dados de Sequência Molecular , Estrutura Molecular , Fator 1 de Elongação de Peptídeos/química , Fator 1 de Elongação de Peptídeos/genética , Interferência de RNA , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMO
Glycosylphosphatidylinositol (GPI) lipids of Trypanosoma brucei undergo lipid remodelling, whereby longer fatty acids on the glycerol are replaced by myristate (C14:0). A similar process occurs on GPI proteins of Saccharomyces cerevisiae where Per1p first deacylates, Gup1p subsequently reacylates the anchor lipid, thus replacing a shorter fatty acid by C26:0. Heterologous expression of the GUP1 homologue of T. brucei in gup1Delta yeast cells partially normalizes the gup1Delta phenotype and restores the transfer of labelled fatty acids from Coenzyme A to lyso-GPI proteins in a newly developed microsomal assay. In this assay, the Gup1p from T. brucei (tbGup1p) strongly prefers C14:0 and C12:0 over C16:0 and C18:0, whereas yeast Gup1p strongly prefers C16:0 and C18:0. This acyl specificity of tbGup1p closely matches the reported specificity of the reacylation of free lyso-GPI lipids in microsomes of T. brucei. Depletion of tbGup1p in trypanosomes by RNAi drastically reduces the rate of myristate incorporation into the sn-2 position of lyso-GPI lipids. Thus, tbGup1p is involved in the addition of myristate to sn-2 during GPI remodelling in T. brucei and can account for the fatty acid specificity of this process. tbGup1p can act on GPI proteins as well as on GPI lipids.
Assuntos
Aciltransferases/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Metabolismo dos Lipídeos , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/enzimologia , Animais , Ácidos Graxos/metabolismo , Glicosilfosfatidilinositóis/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/isolamento & purificação , Microssomos/enzimologia , Ácido Mirístico/metabolismo , Fenótipo , Proteínas de Protozoários/genética , Interferência de RNA , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Trypanosoma brucei brucei/genética , Leveduras/enzimologia , Leveduras/genéticaRESUMO
Phosphatidylethanolamine is a major phospholipid class of all eukaryotic cells. It can be synthesized via the CDP-ethanolamine branch of the Kennedy pathway, by decarboxylation of phosphatidylserine, or by base exchange with phosphatidylserine. The contributions of these pathways to total phosphatidylethanolamine synthesis have remained unclear. Although Trypanosoma brucei, the causative agent of human and animal trypanosomiasis, has served as a model organism to elucidate the entire reaction sequence for glycosylphosphatidylinositol biosynthesis, the pathways for the synthesis of the major phospholipid classes have received little attention. We now show that disruption of the CDP-ethanolamine branch of the Kennedy pathway using RNA interference results in dramatic changes in phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine. By targeting individual enzymes of the pathway, we demonstrate that de novo phosphatidylethanolamine synthesis in T. brucei procyclic forms is strictly dependent on the CDP-ethanolamine route. Interestingly, the last step in the Kennedy pathway can be mediated by two separate activities leading to two distinct pools of phosphatidylethanolamine, consisting of predominantly alk-1-enyl-acyl- or diacyl-type molecular species. In addition, we show that phosphatidylserine in T. brucei procyclic forms is synthesized exclusively by base exchange with phosphatidylethanolamine.
Assuntos
Glicosilfosfatidilinositóis/metabolismo , Modelos Biológicos , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Trypanosoma brucei brucei/enzimologia , Animais , Glicosilfosfatidilinositóis/genética , Humanos , Fosfatidilcolinas/genética , Fosfatidiletanolaminas/genética , Fosfatidilserinas/genética , RNA Interferente Pequeno/genética , Trypanosoma brucei brucei/genética , Tripanossomíase Africana/enzimologia , Tripanossomíase Africana/genéticaRESUMO
Oxidative modifications render low density lipoprotein cytotoxic and enhance its propensity to aggregate and fuse into particles similar to those found in atherosclerotic lesions. We showed previously that aggregation of oxidized LDL (OxLDL) promotes the transformation of human macrophages into lipid-laden foam cells (Asmis, R., and J. Jelk. 2000. Large variations in human foam cell formation in individuals. A fully autologous in vitro assay based on the quantitative analysis of cellular neutral lipids. Atherosclerosis. 148: 243-253). Here, we tested the hypothesis that aggregation of OxLDL enhances its clearance by human macrophages and thus may protect macrophages from OxLDL-induced cytotoxicity. We found that increased aggregation of OxLDL correlated with decreased macrophage injury. Using 3H-labeled and Alexa546-labeled OxLDL, we found that aggregation enhanced OxLDL uptake and increased cholesteryl ester accumulation but did not alter free cholesterol levels in macrophages. Acetylated LDL was a potent competitor of aggregated oxidized LDL (AggOxLDL) uptake, suggesting that scavenger receptor A plays an important role in the clearance of AggOxLDL. Inhibitors of actin polymerization, cytochalasin B, cytochalasin D, and latrunculin A, also prevented AggOxLDL uptake and restored OxLDL-induced cytotoxicity. This suggests that OxLDL-induced macrophage injury does not require OxLDL uptake and may occur on the cell surface. Our data demonstrate that aggregation of cytotoxic OxLDL enhances its clearance by macrophages without damage to the cells, thus allowing macrophages to avoid OxLDL-induced cell injury.