RESUMO
Peroxisomes perform various metabolic processes that are primarily related to the elimination of reactive oxygen species and oxidative lipid metabolism. These organelles are present in all major eukaryotic lineages, nevertheless, information regarding the presence of peroxisomes in opportunistic parasitic protozoa is scarce and in many cases it is still unknown whether these organisms have peroxisomes at all. Here, we performed ultrastructural, cytochemical, and bioinformatic studies to investigate the presence of peroxisomes in three genera of free-living amoebae from two different taxonomic groups that are known to cause fatal infections in humans. By transmission electron microscopy, round structures with a granular content limited by a single membrane were observed in Acanthamoeba castellanii, Acanthamoeba griffini, Acanthamoeba polyphaga, Acanthamoeba royreba, Balamuthia mandrillaris (Amoebozoa), and Naegleria fowleri (Heterolobosea). Further confirmation for the presence of peroxisomes was obtained by treating trophozoites in situ with diaminobenzidine and hydrogen peroxide, which showed positive reaction products for the presence of catalase. We then performed comparative genomic analyses to identify predicted peroxin homologues in these organisms. Our results demonstrate that a complete set of peroxins-which are essential for peroxisome biogenesis, proliferation, and protein import-are present in all of these amoebae. Likewise, our in silico analyses allowed us to identify a complete set of peroxins in Naegleria lovaniensis and three novel peroxin homologues in Naegleria gruberi. Thus, our results indicate that peroxisomes are present in these three genera of free-living amoebae and that they have a similar peroxin complement despite belonging to different evolutionary lineages.
Assuntos
Acanthamoeba castellanii/ultraestrutura , Balamuthia mandrillaris/ultraestrutura , Peroxinas/genética , Peroxissomos/ultraestrutura , Acanthamoeba castellanii/enzimologia , Acanthamoeba castellanii/genética , Balamuthia mandrillaris/enzimologia , Balamuthia mandrillaris/genética , Catalase/metabolismo , Microscopia Eletrônica de Transmissão , Peroxinas/metabolismo , Peroxissomos/enzimologia , Peroxissomos/genética , FilogeniaRESUMO
The protozoan parasite Giardia lamblia has traditionally been reported as lacking peroxisomes, organelles involved in fatty acid metabolism and detoxification of reactive oxygen species. We here report the finding with transmission electron microscopy of an oxidase activity in cytoplasmic vesicles of trophozoites and cysts of G. lamblia. These vesicles were positive to 3,3'-diaminobenzidine and to cerium chloride staining. In addition, using bioinformatic tools, two peroxisomal proteins were identified in the G. lamblia proteome: acyl-CoA synthetase long chain family member 4 (ACSL-4) and peroxin-4 (PEX-4). With confocal and immunoelectron microscopy using polyclonal antibodies both proteins were identified in cytoplasmic vesicles of trophozoites. Altogether, our results suggest for the first time the presence of peroxisomal-like proteins in the cytoplasm of G. lamblia.
Assuntos
Giardia lamblia/química , Peroxissomos/química , Proteínas de Protozoários/isolamento & purificação , 3,3'-Diaminobenzidina/química , Animais , Anticorpos Antiprotozoários/biossíntese , Anticorpos Antiprotozoários/imunologia , Western Blotting , Cério/química , Coenzima A Ligases/imunologia , Coenzima A Ligases/metabolismo , Biologia Computacional , Imunofluorescência , Giardia lamblia/enzimologia , Giardia lamblia/imunologia , Giardia lamblia/ultraestrutura , Histocitoquímica , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Oxirredutases/metabolismo , Peroxinas/análise , Peroxinas/imunologia , Peroxissomos/enzimologia , Proteínas de Protozoários/análise , Coelhos , Coloração e RotulagemRESUMO
Sterol Carrier Protein 2 (SCP2) has been associated with lipid binding and transfer activities. However, genomic, proteomic, and structural studies revealed that it is an ubiquitous domain of complex proteins with a variety functions in all forms of life. High-resolution structures of representative SCP2 domains are available, encouraging a comprehensive review of the structural basis for its success. Most SCP2 domains pertain to three major families and are frequently found as stand-alone or at the C-termini of lipid related peroxisomal enzymes, acetyltransferases causing bacterial resistance, and bacterial environmentally important sulfatases. We (1) analyzed the structural basis of the fold and the classification of SCP2 domains; (2) identified structure-determined sequence features; (3) compared the lipid binding cavity of SCP2 and other lipid binding proteins; (4) surveyed proposed mechanisms of SCP2 mediated lipid transfer between membranes; and (5) uncovered a possible new function of the SCP2 domain as a protein-protein recognition device.
Assuntos
Proteínas de Transporte/química , Lipídeos/química , Esteróis/química , Proteínas de Transporte/metabolismo , Humanos , Peroxissomos/enzimologia , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Mapas de Interação de Proteínas , ProteômicaRESUMO
SCOPE: Omega-3 polyunsaturated fatty acids (n-3 PUFA) found in fish oil activate PPAR-α, stimulate peroxisomal fatty acid (FA) ß-oxidation and prevent impairments on glucose homeostasis. METHODS AND RESULTS: Glucose metabolism and FA oxidation were studied in C57/Bl6 mice fed with diets containing either 3.6 and 31.5% fish oil or lard. To assess the effects of peroxisomal proliferation on FA oxidation independent of n-3 PUFA intake, mice were treated with the PPAR-α agonist WY-14643. n-3 PUFA-fed mice were protected from glucose intolerance and dyslipidemia compared to animals fed a lard-based high-fat diet. Most importantly, mice fed on the hyperlipidic diet based on fish oil as well as the WY-14643 treated mice showed twofold increase of odd, medium-chain, dicarboxylic acylcarnitines in the liver suggesting that not only ß-oxidation, but also α- and ω-oxidation of FA were increased. Finally, an oxidation assay using liver homogenates and palmitic acid as substrate revealed an over tenfold increased production of similar acylcarnitines, indicating that FA are their precursors. CONCLUSION: This study shows at the metabolite level that peroxisome proliferation induced either by fish oil or WY-14643 is associated with increased α- and ω-oxidation of FA producing specific acylcarnitines that can be utilized as biomarkers of peroxisomal FA oxidation.
Assuntos
Carnitina/análogos & derivados , Dieta Hiperlipídica/efeitos adversos , Gorduras Insaturadas na Dieta/metabolismo , Ácidos Graxos Ômega-3/metabolismo , Fígado/metabolismo , Sobrepeso/metabolismo , Peroxissomos/metabolismo , Animais , Biomarcadores/química , Biomarcadores/metabolismo , Carnitina/química , Carnitina/metabolismo , Gorduras na Dieta/efeitos adversos , Gorduras Insaturadas na Dieta/efeitos adversos , Gorduras Insaturadas na Dieta/uso terapêutico , Ácidos Graxos Ômega-3/uso terapêutico , Óleos de Peixe/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Intolerância à Glucose/etiologia , Intolerância à Glucose/prevenção & controle , Hiperlipidemias/etiologia , Hiperlipidemias/prevenção & controle , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Camundongos Endogâmicos C57BL , Peso Molecular , Sobrepeso/etiologia , Sobrepeso/fisiopatologia , Sobrepeso/prevenção & controle , Oxirredução , Proliferadores de Peroxissomos/farmacologia , Peroxissomos/efeitos dos fármacos , Peroxissomos/enzimologia , Pirimidinas/farmacologiaRESUMO
The physiological role of peroxisomal ascorbate peroxidases (pAPX) is unknown; therefore, we utilized pAPX4 knockdown rice and catalase (CAT) inhibition to assess its role in CAT compensation under high photorespiration. pAPX4 knockdown induced co-suppression in the expression of pAPX3. The rice mutants exhibited metabolic changes such as lower CAT and glycolate oxidase (GO) activities and reduced glyoxylate content; however, APX activity was not altered. CAT inhibition triggered different changes in the expression of CAT, APX and glutathione peroxidase (GPX) isoforms between non-transformed (NT) and silenced plants. These responses were associated with alterations in APX, GPX and GO activities, suggesting redox homeostasis differences. The glutathione oxidation-reduction states were modulated differently in mutants, and the ascorbate redox state was greatly affected in both genotypes. The pAPX suffered less oxidative stress and photosystem II (PSII) damage and displayed higher photosynthesis than the NT plants. The improved acclimation exhibited by the pAPX plants was indicated by lower H2 O2 accumulation, which was associated with lower GO activity and glyoxylate content. The suppression of both pAPXs and/or its downstream metabolic and molecular effects may trigger favourable antioxidant and compensatory mechanisms to cope with CAT deficiency. This physiological acclimation may involve signalling by peroxisomal H2 O2 , which minimized the photorespiration.
Assuntos
Antioxidantes/metabolismo , Ascorbato Peroxidases/genética , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/metabolismo , Oryza/fisiologia , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Ascorbato Peroxidases/metabolismo , Catalase/genética , Catalase/metabolismo , Respiração Celular , Técnicas de Silenciamento de Genes , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Oryza/enzimologia , Oryza/genética , Oryza/efeitos da radiação , Oxirredução , Estresse Oxidativo , Peroxissomos/enzimologia , Fotossíntese , Complexo de Proteína do Fotossistema II/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente ModificadasRESUMO
Estrogen deficiency accelerates the development of several disorders including visceral obesity and hepatic steatosis. The predisposing factors can be exacerbated by drugs that affect hepatic lipid metabolism. The aim of the present work was to determine if raloxifene, a selective estrogen receptor modulator (SERM) used extensively by postmenopausal women, affects hepatic fatty acid oxidation pathways. Fatty acids oxidation was measured in the livers, mitochondria and peroxisomes of ovariectomized (OVX) rats. Mitochondrial and peroxisomal ß-oxidation was inhibited by raloxifene at a concentration range of 2.5-25 µM. In perfused livers, raloxifene reduced the ketogenesis from endogenous and exogenous fatty acids and increased the ß-hydroxybutyrate/acetoacetate ratio. An increase in ¹4CO2 production without a parallel increase in the oxygen consumption indicated that raloxifene caused a diversion of NADH from the mitochondrial respiratory chain to another oxidative reaction. It was found that raloxifene has a strong ability to react with H2O2 in the presence of peroxidase. It is likely that the generation of phenoxyl radical derivatives of raloxifene in intact livers led to the co-oxidation of NADH and a shift of the cellular redox state to an oxidised condition. This change can perturb other important liver metabolic processes dependent on cellular NADH/NAD⺠ratio.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Ácidos Graxos/metabolismo , Fígado Gorduroso/induzido quimicamente , Fígado/efeitos dos fármacos , Oxidantes/efeitos adversos , Cloridrato de Raloxifeno/efeitos adversos , Moduladores Seletivos de Receptor Estrogênico/efeitos adversos , Acil Coenzima A/metabolismo , Acil-CoA Oxidase/metabolismo , Animais , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Progressão da Doença , Terapia de Reposição de Estrogênios/efeitos adversos , Fígado Gorduroso/metabolismo , Fígado Gorduroso/fisiopatologia , Feminino , Peróxido de Hidrogênio/química , Fígado/enzimologia , Fígado/metabolismo , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Hepáticas/metabolismo , Ovariectomia/efeitos adversos , Oxidantes/química , Oxirredução , Peroxidase/metabolismo , Peroxissomos/efeitos dos fármacos , Peroxissomos/enzimologia , Peroxissomos/metabolismo , Cloridrato de Raloxifeno/química , Ratos , Moduladores Seletivos de Receptor Estrogênico/químicaRESUMO
The aim of this work was to evaluate the effects of therapeutic doses of Cimicifuga racemosa on cardiovascular parameters and on liver lipid metabolism and redox status in an animal model of estrogen deficiency associated with hypertension, a condition that could make the liver more vulnerable to drug-induced injuries. Female Wistar rats were subjected to the surgical procedures of bilateral ovariectomy (OVX) and induction of renovascular hypertension (two-kidneys, one-clip; 2K1C). These animals (OVX + 2K1C) were treated with daily doses of a C. racemosa extract, using a dose that is similar to that recommended to postmenopausal women (0.6 mg/kg), over a period of 15 days. The results were compared to those of untreated OVX + 2K1C, OVX, and control rats. The treatment with C. racemosa caused a significant reduction in blood pressure. In the liver, treatment did not prevent the development of steatosis, and it reduced the mitochondrial and peroxisomal capacity to oxidize octanoyl-CoA compared to the untreated animals. In addition, C. racemosa caused numerous undesirable effects on the liver redox status: it increased the mitochondrial reactive oxygen species generation, an event that was not accompanied by an increase in the activity of superoxide dismutase, and it induced a decrease in peroxisomal catalase activity. Although the reduced glutathione content had not been affected, a phenomenon that probably reflected the restoration of glucose-6-phosphate dehydrogenase activity by C. racemosa, oxidative damage was evidenced by the elevated level of thiobarbituric acid-reactive substances found in the liver of treated animals.
Assuntos
Anti-Hipertensivos/farmacologia , Cimicifuga/química , Ácidos Graxos/metabolismo , Hipertensão Renovascular/metabolismo , Fígado/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Acil-CoA Oxidase/metabolismo , Animais , Catalase/metabolismo , Estrogênios/deficiência , Fígado Gorduroso/sangue , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/metabolismo , Feminino , Hipertensão Renovascular/sangue , Hipertensão Renovascular/tratamento farmacológico , Metabolismo dos Lipídeos , Lipídeos/sangue , Fígado/enzimologia , Fígado/metabolismo , Mitocôndrias Hepáticas/metabolismo , Ovariectomia , Oxirredução , Consumo de Oxigênio , Peroxissomos/enzimologia , Peroxissomos/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
Adenylate kinases are key enzymes involved in cell energy management. Trypanosomatid organisms have the largest number of isoforms found in a single cell, constituting a major difference with the mammalian hosts. In this work we study an adenylate kinase, TcADK3, the only Trypanosoma cruzi protein harboring the putative peroxisomal (glycosomal) targeting signal, "-CKL". Parasites expressing GFP fused to TcADK3 showed a strong fluorescence in the glycosomes. The same result was obtained when the tripeptide "-CKL" was added at the C-terminus of the GFP, demonstrating that this signal is necessary and sufficient for targeting proteins to glycosomes. When this tripeptide was removed from the GFP-TcADK3 fusion protein, the fluorescence was re-localized in the cytoplasm. The CKL signal could be used for targeting foreign proteins to the glycosomes. This model also provides a useful tool to study glycosomes dynamics, morphology or number in living parasites in any stage of the life cycle.
Assuntos
Adenilato Quinase/metabolismo , Microcorpos/enzimologia , Peroxissomos/enzimologia , Transdução de Sinais , Trypanosoma cruzi/enzimologia , Adenilato Quinase/química , Adenilato Quinase/genética , Sequência de Aminoácidos , Clonagem Molecular , Regulação Enzimológica da Expressão Gênica , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Microscopia de Fluorescência , Plasmídeos , Transfecção , Trypanosoma cruzi/genéticaRESUMO
Transketolase has been characterized in Leishmania mexicana. A gene encoding this enzyme was identified and cloned. The gene was expressed in Escherichia coli and the protein was purified and characterized. An apparent K(m) of 2.75 mM for ribose 5-phosphate was determined. X-ray crystallography was used to determine the three-dimensional structure of the enzyme to a resolution of 2.2 A (1 A identical with 0.1 nm). The C-terminus of the protein contains a type-1 peroxisome-targeting signal, suggestive of a possible glycosomal subcellular localization. Subcellular localization experiments performed with promastigote forms of the parasite revealed that the protein was predominantly cytosolic, although a significant component of the total activity was associated with the glycosomes. Transketolase is thus the first enzyme of the nonoxidative branch of the pentose phosphate pathway whose presence has been demonstrated in a peroxisome-like organelle.
Assuntos
Leishmania mexicana/química , Leishmania mexicana/enzimologia , Transcetolase/metabolismo , Sequência de Aminoácidos/genética , Animais , Clonagem Molecular , Cristalografia por Raios X/métodos , DNA de Protozoário/genética , Leishmania mexicana/crescimento & desenvolvimento , Microcorpos/química , Microcorpos/enzimologia , Dados de Sequência Molecular , Peroxissomos/química , Peroxissomos/enzimologia , Sinais Direcionadores de Proteínas/fisiologia , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Transcetolase/biossíntese , Transcetolase/química , Transcetolase/genéticaRESUMO
Oxidized low density lipoprotein (LDL) has an important proinflammatory role in atherogenesis. In this study, we investigated the ability of oxidized LDL (oxLDL) and its phospholipid components to induce lipid body formation in leukocytes. Incubation of mouse peritoneal macrophages with oxidized, but not with native LDL led to lipid body formation within 1 h. This was blocked by platelet-activating factor (PAF) receptor antagonists or by preincubation of oxLDL with rPAF acetylhydrolase. HPLC fractions of phospholipids purified from oxLDL induced calcium flux in neutrophils as well as lipid body formation in macrophages. Injection of the bioactive phospholipid fractions or butanoyl and butenoyl PAF, a phospholipid previously shown to be present in oxLDL, into the pleural cavity of mice induced lipid body formation in leukocytes recovered after 3 h. The 5-lipoxygenase and cyclooxygenase-2 colocalized within lipid bodies formed after stimulation with oxLDL, bioactive phospholipid fractions, or butanoyl and butenoyl PAF. Lipid body formation was inhibited by 5-lipoxygenase antagonists, but not by cyclooxygenase-2 inhibitors. Azelaoyl-phosphatidylcholine, a peroxisome proliferator-activated receptor-gamma agonist in oxLDL phospholipid fractions, induced formation of lipid bodies at late time points (6 h) and synergized with suboptimal concentrations of oxLDL. We conclude that lipid body formation is an important proinflammatory effect of oxLDL and that PAF-like phospholipids and peroxisome proliferator-activated receptor-gamma agonists generated during LDL oxidation are important mediators in this phenomenon.
Assuntos
Corpos de Inclusão/metabolismo , Leucócitos/metabolismo , Lipoproteínas LDL/metabolismo , Peroxissomos/metabolismo , Fator de Ativação de Plaquetas/fisiologia , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/fisiologia , Fatores de Transcrição/agonistas , Fatores de Transcrição/fisiologia , Animais , Araquidonato 5-Lipoxigenase/metabolismo , Araquidonato 5-Lipoxigenase/fisiologia , Ciclo-Oxigenase 2 , Sinergismo Farmacológico , Feminino , Humanos , Isoenzimas/metabolismo , Leucócitos/fisiologia , Lipoproteínas LDL/administração & dosagem , Lipoproteínas LDL/farmacologia , Macrófagos Peritoneais/metabolismo , Masculino , Proteínas de Membrana , Camundongos , Oxirredução , Peroxissomos/enzimologia , Peroxissomos/fisiologia , Fosfolipídeos/metabolismo , Fator de Ativação de Plaquetas/administração & dosagem , Fator de Ativação de Plaquetas/metabolismo , Cavidade Pleural/citologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Cavidade Torácica , Fatores de Transcrição/metabolismoRESUMO
We demonstrated a neutral Mg-ATPase activity in human peroxisomal membranes. To establish the precise experimental conditions for detection of this ATPase, both cytochemical and biochemical characterizations were first carried out in liver peroxisomes from control and cipofibrate-treated rats. The results demonstrated an Mg-ATPase reaction in both normal and proliferated peroxisomes. The nucleotidase activity, with marked preference for ATP, was sensitive to the inhibitors N-ethylmaleimide and 7-chloro-4-nitro-benzo-2-oxadiazole (NBDCl). An ultrastructural cytochemical analysis was developed to evaluate the peroxisomal localization, which localized the reaction product to the peroxisomal membrane. These characteristics can help to differentiate the peroxisomal ATPase from the activity found in mitochondria and endoplasmic reticulum. The conditions established for detecting the rat peroxisomal ATPase were then applied to human peroxisomes isolated from liver and skin fibroblasts in culture. A similar Mg-ATPase activity was readily shown, both cytochemically and biochemically, in the membranes of human peroxisomes. These results, together with previous evidence, strongly support the presence of a specific ATPase in the human peroxisomal membrane. This ATPase may play a crucial role in peroxisome biogenesis.
Assuntos
ATPase de Ca(2+) e Mg(2+)/análise , Histocitoquímica , Membranas Intracelulares/enzimologia , Peroxissomos/enzimologia , Trifosfato de Adenosina/metabolismo , Animais , ATPase de Ca(2+) e Mg(2+)/antagonistas & inibidores , ATPase de Ca(2+) e Mg(2+)/metabolismo , Ácido Clofíbrico/análogos & derivados , Ácido Clofíbrico/farmacologia , Retículo Endoplasmático/enzimologia , Inibidores Enzimáticos/farmacologia , Etilmaleimida/farmacologia , Ácidos Fíbricos , Fibroblastos/enzimologia , Humanos , Fígado/ultraestrutura , Mitocôndrias/enzimologia , Proliferadores de Peroxissomos/farmacologia , Ratos , Ratos Sprague-Dawley , Pele/ultraestrutura , Especificidade por SubstratoRESUMO
Three Japanese patients with peroxisomal acyl coenzyme A oxidase deficiency who manifested psychomotor retardation and regression during the late infantile period showed characteristic patterns of demyelination in the ponto- medullary corticospinal tracts and in the cerebellar and cerebral white matter. Molecular investigations revealed 2 novel missense mutations, M278V and G178C.