RESUMO
During Mycobacterium tuberculosis (Mtb) infection, the virulence factor PtpA belonging to the protein tyrosine phosphatase family is delivered into the cytosol of the macrophage. PtpA interacts with numerous eukaryotic proteins modulating phagosome maturation, innate immune response, apoptosis, and potentially host-lipid metabolism, as previously reported by our group. In vitro, the human trifunctional protein enzyme (hTFP) is a bona fide PtpA substrate, a key enzyme of mitochondrial ß-oxidation of long-chain fatty acids, containing two alpha and two beta subunits arranged in a tetramer structure. Interestingly, it has been described that the alpha subunit of hTFP (ECHA, hTFPα) is no longer detected in mitochondria during macrophage infection with the virulent Mtb H37Rv. To better understand if PtpA could be the bacterial factor responsible for this effect, in the present work, we studied in-depth the PtpA activity and interaction with hTFPα. With this aim, we performed docking and in vitro dephosphorylation assays defining the P-Tyr-271 as the potential target of mycobacterial PtpA, a residue located in the helix-10 of hTFPα, previously described as relevant for its mitochondrial membrane localization and activity. Phylogenetic analysis showed that Tyr-271 is absent in TFPα of bacteria and is present in more complex eukaryotic organisms. These results suggest that this residue is a specific PtpA target, and its phosphorylation state is a way of regulating its subcellular localization. We also showed that phosphorylation of Tyr-271 can be catalyzed by Jak kinase. In addition, we found by molecular dynamics that PtpA and hTFPα form a stable protein complex through the PtpA active site, and we determined the dissociation equilibrium constant. Finally, a detailed study of PtpA interaction with ubiquitin, a reported PtpA activator, showed that additional factors are required to explain a ubiquitin-mediated activation of PtpA. Altogether, our results provide further evidence supporting that PtpA could be the bacterial factor that dephosphorylates hTFPα during infection, potentially affecting its mitochondrial localization or ß-oxidation activity.
Assuntos
Proteínas de Bactérias , Proteína Mitocondrial Trifuncional , Mycobacterium tuberculosis , Humanos , Metabolismo dos Lipídeos , Filogenia , Ubiquitinas , Proteína Mitocondrial Trifuncional/metabolismo , Proteínas de Bactérias/metabolismoRESUMO
The pathogenesis of the muscular symptoms and recurrent rhabdomyolysis that are commonly manifested in patients with mitochondrial trifunctional protein (MTP) and long-chain 3-hydroxy-acyl-CoA dehydrogenase (LCHAD) deficiencies is still unknown. In this study we investigated the effects of the major long-chain monocarboxylic 3-hydroxylated fatty acids (LCHFA) accumulating in these disorders, namely 3-hydroxytetradecanoic (3HTA) and 3-hydroxypalmitic (3HPA) acids, on important mitochondrial functions in rat skeletal muscle mitochondria. 3HTA and 3HPA markedly increased resting (state 4) and decreased ADP-stimulated (state 3) and CCCP-stimulated (uncoupled) respiration. 3HPA provoked similar effects in permeabilized skeletal muscle fibers, validating the results obtained in purified mitochondria. Furthermore, 3HTA and 3HPA markedly diminished mitochondrial membrane potential, NAD(P)H content and Ca(2+) retention capacity in Ca(2+)-loaded mitochondria. Mitochondrial permeability transition (mPT) induction probably underlie these effects since they were totally prevented by cyclosporin A and ADP. In contrast, the dicarboxylic analogue of 3HTA did not alter the tested parameters. Our data strongly indicate that 3HTA and 3HPA behave as metabolic inhibitors, uncouplers of oxidative phosphorylation and mPT inducers in skeletal muscle. It is proposed that these pathomechanisms disrupting mitochondrial homeostasis may be involved in the muscle alterations characteristic of MTP and LCHAD deficiencies.
Assuntos
Cardiomiopatias/metabolismo , Erros Inatos do Metabolismo Lipídico/metabolismo , Mitocôndrias Musculares/efeitos dos fármacos , Miopatias Mitocondriais/metabolismo , Proteína Mitocondrial Trifuncional/deficiência , Ácidos Mirísticos/farmacologia , Doenças do Sistema Nervoso/metabolismo , Ácidos Palmíticos/farmacologia , Rabdomiólise/metabolismo , Animais , Cálcio/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/fisiologia , Proteína Mitocondrial Trifuncional/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , NADP/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Ratos WistarRESUMO
Mitochondrial trifunctional protein and long-chain 3-hydroxyacyl-CoA dehydrogenase deficiencies are fatty acid oxidation disorders biochemically characterized by tissue accumulation of long-chain fatty acids and derivatives, including the monocarboxylic long-chain 3-hydroxy fatty acids (LCHFAs) 3-hydroxytetradecanoic acid (3HTA) and 3-hydroxypalmitic acid (3HPA). Patients commonly present severe cardiomyopathy for which the pathogenesis is still poorly established. We investigated the effects of 3HTA and 3HPA, the major metabolites accumulating in these disorders, on important parameters of mitochondrial homeostasis in Ca(2+) -loaded heart mitochondria. 3HTA and 3HPA significantly decreased mitochondrial membrane potential, the matrix NAD(P)H pool and Ca(2+) retention capacity, and also induced mitochondrial swelling. These fatty acids also provoked a marked decrease of ATP production reflecting severe energy dysfunction. Furthermore, 3HTA-induced mitochondrial alterations were completely prevented by the classical mitochondrial permeability transition (mPT) inhibitors cyclosporin A and ADP, as well as by ruthenium red, a Ca(2+) uptake blocker, indicating that LCHFAs induced Ca(2+)-dependent mPT pore opening. Milder effects only achieved at higher doses of LCHFAs were observed in brain mitochondria, implying a higher vulnerability of heart to these fatty acids. By contrast, 3HTA and docosanoic acids did not change mitochondrial homeostasis, indicating selective effects for monocarboxylic LCHFAs. The present data indicate that the major LCHFAs accumulating in mitochondrial trifunctional protein and long-chain 3-hydroxyacyl-CoA dehydrogenase deficiencies induce mPT pore opening, compromising Ca(2+) homeostasis and oxidative phosphorylation more intensely in the heart. It is proposed that these pathomechanisms may contribute at least in part to the severe cardiac alterations characteristic of patients affected by these diseases.
Assuntos
Sinalização do Cálcio , 3-Hidroxiacil-CoA Desidrogenase de Cadeia Longa/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Ácidos Mirísticos/metabolismo , Fosforilação Oxidativa , Ácidos Palmíticos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Cardiomiopatias/enzimologia , Cardiomiopatias/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Erros Inatos do Metabolismo Lipídico/enzimologia , Erros Inatos do Metabolismo Lipídico/metabolismo , 3-Hidroxiacil-CoA Desidrogenase de Cadeia Longa/deficiência , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/enzimologia , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Miopatias Mitocondriais/enzimologia , Miopatias Mitocondriais/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Dilatação Mitocondrial/efeitos dos fármacos , Proteína Mitocondrial Trifuncional/deficiência , Proteína Mitocondrial Trifuncional/metabolismo , NADP/metabolismo , Doenças do Sistema Nervoso/enzimologia , Doenças do Sistema Nervoso/metabolismo , Especificidade de Órgãos , Fosforilação Oxidativa/efeitos dos fármacos , Ratos Wistar , Rabdomiólise/enzimologia , Rabdomiólise/metabolismoRESUMO
Patients with long-chain 3-hydroxy-acyl-CoA dehydrogenase (LCHAD) deficiency commonly present liver dysfunction whose pathogenesis is unknown. We studied the effects of long-chain 3-hydroxylated fatty acids (LCHFA) that accumulate in LCHAD deficiency on liver bioenergetics using mitochondrial preparations from young rats. We provide strong evidence that 3-hydroxytetradecanoic (3HTA) and 3-hydroxypalmitic (3HPA) acids, the monocarboxylic acids that are found at the highest tissue concentrations in this disorder, act as metabolic inhibitors and uncouplers of oxidative phosphorylation. These conclusions are based on the findings that these fatty acids decreased ADP-stimulated (state 3) and uncoupled respiration, mitochondrial membrane potential and NAD(P)H content, and, in contrast, increased resting (state 4) respiration. We also verified that 3HTA and 3HPA markedly reduced Ca2+ retention capacity and induced swelling in Ca2+-loaded mitochondria. These effects were mediated by mitochondrial permeability transition (MPT) induction since they were totally prevented by the classical MPT inhibitors cyclosporin A and ADP, as well as by ruthenium red, a Ca2+ uptake blocker. Taken together, our data demonstrate that the major monocarboxylic LCHFA accumulating in LCHAD deficiency disrupt energy mitochondrial homeostasis in the liver. It is proposed that this pathomechanism may explain at least in part the hepatic alterations characteristic of the affected patients.
Assuntos
3-Hidroxiacil-CoA Desidrogenases/deficiência , Cardiomiopatias/patologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Ácidos Graxos/farmacologia , Erros Inatos do Metabolismo Lipídico/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Miopatias Mitocondriais/patologia , Dilatação Mitocondrial/efeitos dos fármacos , Doenças do Sistema Nervoso/patologia , Rabdomiólise/patologia , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Animais , Transporte Biológico , Cálcio/metabolismo , Cardiomiopatias/metabolismo , Erros Inatos do Metabolismo Lipídico/metabolismo , Mitocôndrias Hepáticas/metabolismo , Membranas Mitocondriais/metabolismo , Miopatias Mitocondriais/metabolismo , Proteína Mitocondrial Trifuncional/deficiência , NADP/metabolismo , Doenças do Sistema Nervoso/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Ratos Wistar , Rabdomiólise/metabolismoRESUMO
Cardiomyopathy is a common clinical feature of some inherited disorders of mitochondrial fatty acid ß-oxidation including mitochondrial trifunctional protein (MTP) and isolated long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiencies. Since individuals affected by these disorders present tissue accumulation of various fatty acids, including long-chain 3-hydroxy fatty acids, in the present study we investigated the effect of 3-hydroxydecanoic (3 HDCA), 3-hydroxydodecanoic (3 HDDA), 3-hydroxytetradecanoic (3 HTA) and 3-hydroxypalmitic (3 HPA) acids on mitochondrial oxidative metabolism, estimated by oximetry, NAD(P)H content, hydrogen peroxide production, membrane potential (ΔΨ) and swelling in rat heart mitochondrial preparations. We observed that 3 HTA and 3 HPA increased resting respiration and diminished the respiratory control and ADP/O ratios using glutamate/malate or succinate as substrates. Furthermore, 3 HDDA, 3 HTA and 3 HPA decreased ΔΨ, the matrix NAD(P)H pool and hydrogen peroxide production. These data indicate that these fatty acids behave as uncouplers of oxidative phosphorylation. We also verified that 3 HTA-induced uncoupling-effect was not mediated by the adenine nucleotide translocator and that this fatty acid induced the mitochondrial permeability transition pore opening in calcium-loaded organelles since cyclosporin A prevented the reduction of mitochondrial ΔΨ and swelling provoked by 3 HTA. The present data indicate that major 3-hydroxylated fatty acids accumulating in MTP and LCHAD deficiencies behave as strong uncouplers of oxidative phosphorylation potentially impairing heart energy homeostasis.
Assuntos
3-Hidroxiacil-CoA Desidrogenases/metabolismo , Cardiomiopatias/metabolismo , Ácidos Graxos/metabolismo , Erros Inatos do Metabolismo Lipídico/metabolismo , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Fosforilação Oxidativa , Doenças do Sistema Nervoso Periférico/metabolismo , Retinose Pigmentar/metabolismo , 3-Hidroxiacil-CoA Desidrogenases/deficiência , Animais , Peróxido de Hidrogênio/metabolismo , 3-Hidroxiacil-CoA Desidrogenase de Cadeia Longa , Miopatias Mitocondriais , Proteína Mitocondrial Trifuncional/deficiência , Doenças do Sistema Nervoso , Oxirredução , Consumo de Oxigênio , Ratos , Ratos Wistar , RabdomióliseRESUMO
Accumulation of long-chain 3-hydroxy fatty acids is the biochemical hallmark of long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) and mitochondrial trifunctional protein (MTP) deficiencies. These disorders are clinically characterized by neurological symptoms, such as convulsions and lethargy, as well as by cardiomyopathy and muscle weakness. In the present work we investigated the in vitro effect of 3-hydroxydodecanoic (3HDA), 3-hydroxytetradecanoic (3HTA) and 3-hydroxypalmitic (3HPA) acids, which accumulate in these disorders, on important oxidative stress parameters in cerebral cortex of young rats in the hope to clarify the mechanisms leading to the brain damage found in patients affected by these disorders. It was first verified that these compounds significantly induced lipid peroxidation, as determined by increased thiobarbituric acid-reactive substances levels. In addition, carbonyl formation was significantly increased and sulfhydryl content decreased by 3HTA and 3HPA, which indicates that these fatty acids elicit protein oxidative damage. 3HTA and 3HPA also diminished the reduced glutathione (GSH) levels, without affecting nitrate and nitrite production. Finally, we observed that the addition of the antioxidants and free radical scavengers trolox and deferoxamine (DFO) was able to partially prevent lipid oxidative damage, whereas DFO fully prevented the reduction on GSH levels induced by 3HTA. Our present data showing that 3HDA, 3HTA and 3HPA elicit oxidative stress in rat brain indicate that oxidative damage may represent an important pathomechanism involved in the neurologic symptoms manifested by patients affected by LCHAD and MTP deficiencies.
Assuntos
3-Hidroxiacil-CoA Desidrogenases/deficiência , Encefalopatias Metabólicas/metabolismo , Encéfalo/metabolismo , Transtornos do Metabolismo dos Lipídeos/metabolismo , Complexos Multienzimáticos/deficiência , Ácidos Mirísticos/toxicidade , Estresse Oxidativo/fisiologia , Ácidos Palmíticos/toxicidade , Animais , Encéfalo/efeitos dos fármacos , Encefalopatias Metabólicas/induzido quimicamente , Ácidos Decanoicos/metabolismo , Ácidos Decanoicos/toxicidade , Ácidos Graxos/metabolismo , Ácidos Graxos/toxicidade , Transtornos do Metabolismo dos Lipídeos/induzido quimicamente , 3-Hidroxiacil-CoA Desidrogenase de Cadeia Longa , Masculino , Proteína Mitocondrial Trifuncional , Ácidos Mirísticos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ácidos Palmíticos/metabolismo , Ratos , Ratos WistarRESUMO
AIMS: We investigated the in vitro effects of 3-hydroxydodecanoic (3HDA), 3-hydroxytetradecanoic (3HTA) and 3-hydroxypalmitic (3HPA) acids, which accumulate in tissues of patients affected by mitochondrial trifunctional protein (MTP) and isolated long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiencies, on various parameters of energy homeostasis in mitochondrial preparations from brain of young rats. MAIN METHODS: We measured the respiratory parameters state 4, state 3, respiratory control ratio (RCR) and ADP/O ratio by the rate of oxygen consumption, as well as the mitochondrial membrane potential and the matrix NAD(P)H levels in the presence of the fatty acids. KEY FINDINGS: We found that 3HDA, 3HTA and 3HPA markedly increased state 4 respiration and diminished the RCR using glutamate plus malate or succinate as substrates. 3HTA and 3HPA also diminished the mitochondrial membrane potential and the matrix NAD(P)H levels. In addition, 3HTA decreased state 3 respiration using glutamate/malate, but not pyruvate/malate or succinate as substrates. Our data indicate that the long-chain 3-hydroxy fatty acids that accumulate in LCHAD/MTP deficiencies act as uncouplers of oxidative phosphorylation, while 3HTA also behaves as a metabolic inhibitor. SIGNIFICANCE: It is presumed that impairment of brain energy homeostasis caused by these endogenous accumulating compounds may contribute at least in part to the neuropathology of LCHAD/MTP deficiencies.
Assuntos
3-Hidroxiacil-CoA Desidrogenases/deficiência , Encéfalo/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Ácidos Láuricos/farmacologia , Mitocôndrias/efeitos dos fármacos , Complexos Multienzimáticos/deficiência , Ácidos Mirísticos/farmacologia , 3-Hidroxiacil-CoA Desidrogenases/efeitos dos fármacos , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Animais , Encéfalo/metabolismo , Homeostase/efeitos dos fármacos , 3-Hidroxiacil-CoA Desidrogenase de Cadeia Longa , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteína Mitocondrial Trifuncional , Complexos Multienzimáticos/efeitos dos fármacos , Complexos Multienzimáticos/metabolismo , NADP/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
OBJECTIVE: To determine the spectrum of presentation, including both clinical and biochemical abnormalities, and the clinical course in a cohort of patients with complete mitochondrial trifunctional protein (MTP) deficiency, a rare inborn error of mitochondrial fatty acid oxidation. STUDY DESIGN: A questionnaire was sent to the referring physicians from 25 unselected MTP-deficient patients. RESULTS: Twenty-one patients could be included. Questionnaires about four patients were not returned. Nine (43%) patients presented with rapidly progressive clinical deterioration; six (67%) of them had hypoketotic hypoglycemia. The remaining 12 patients presented with a much more insidious disease with nonspecific chronic symptoms, including hypotonia (100%), cardiomyopathy (73%), failure to thrive, or peripheral neuropathy. Ten patients (48%) presented in the neonatal period. Mortality was high (76%), mostly attributable to cardiac involvement. Two patients who were diagnosed prenatally died despite treatment. CONCLUSION: Complete MTP deficiency often presents with nonspecific symptomatology, which makes clinical recognition difficult. Hypotonia and cardiomyopathy are common presenting features, and the differential diagnosis of an infant with these signs should include MTP deficiency. In spite of early diagnosis and treatment, only a few patients with this condition have survived.
Assuntos
Erros Inatos do Metabolismo Lipídico/diagnóstico , Complexos Multienzimáticos/deficiência , Cardiomiopatias/etiologia , Cardiomiopatias/metabolismo , Progressão da Doença , Feminino , Cardiopatias/etiologia , Cardiopatias/metabolismo , Humanos , Lactente , Recém-Nascido , Masculino , Proteína Mitocondrial Trifuncional , Hipotonia Muscular/etiologia , Hipotonia Muscular/metabolismoRESUMO
OBJECTIVES: To evaluate the feasibility of molecular prenatal diagnosis in families with mitochondrial trifunctional protein (TFP) mutations and prospectively study the effects of fetal genotype on pregnancy outcome. TFP catalyzes the last 3 steps in mitochondrial long-chain fatty acid oxidation. STUDY DESIGN: We performed molecular prenatal diagnosis in 9 pregnancies, 8 in 6 families with isolated long-chain 3-hydroxyacyl-coenzyme A dehydrogenase (LCHAD) deficiency and one in a family with complete TFP deficiency. Analyses were performed on chorionic villous samples in 7 pregnancies and on amniocytes in 2. RESULTS: Molecular prenatal diagnosis successfully identified the fetal genotype in all 9 pregnancies. Two fetuses were affected, and both pregnancies were terminated by family decision. Two other fetuses had normal genotype and 5 others were heterozygotes. These 7 pregnancies were uncomplicated, and all the offspring are alive and apparently healthy. Genotypes of the aborted fetuses and neonates were confirmed by molecular analysis and enzymatic assays. CONCLUSIONS: Molecular prenatal diagnosis is possible and valid in guiding management of pregnancies in families with known TFP defects. Women heterozygous for TFP alpha-subunit mutations who carry fetuses with wild-type or heterozygous genotypes have uncomplicated pregnancies.
Assuntos
Amniocentese , Amostra da Vilosidade Coriônica , Erros Inatos do Metabolismo Lipídico/diagnóstico , Complexos Multienzimáticos/deficiência , Palmitoil-CoA Hidrolase/deficiência , Fígado Gorduroso/etiologia , Feminino , Genótipo , Síndrome HELLP/etiologia , Humanos , Erros Inatos do Metabolismo Lipídico/genética , Proteína Mitocondrial Trifuncional , Complexos Multienzimáticos/genética , Palmitoil-CoA Hidrolase/genética , Gravidez , Complicações na Gravidez/etiologia , Resultado da GravidezRESUMO
Mitochondrial trifunctional protein deficiency, a recently identified disorder of fatty-acid oxidation, may show characteristic features such as peripheral neuropathy, pigmentary retinopathy, and acute fatty liver degeneration in pregnant women with an affected fetus. We describe a patient with trifunctional protein deficiency whose clinical picture consisted of severe calcium and phosphate abnormalities caused by hypoparathyroidism.