RESUMO
Clear cell renal cell carcinoma (ccRCC) is histologically defined by its lipid and glycogen-rich cytoplasmic deposits. Alterations in the VHL tumor suppressor stabilizing the hypoxia-inducible factors (HIFs) are the most prevalent molecular features of clear cell tumors. The significance of lipid deposition remains undefined. We describe the mechanism of lipid deposition in ccRCC by identifying the rate-limiting component of mitochondrial fatty acid transport, carnitine palmitoyltransferase 1A (CPT1A), as a direct HIF target gene. CPT1A is repressed by HIF1 and HIF2, reducing fatty acid transport into the mitochondria, and forcing fatty acids to lipid droplets for storage. Droplet formation occurs independent of lipid source, but only when CPT1A is repressed. Functionally, repression of CPT1A is critical for tumor formation, as elevated CPT1A expression limits tumor growth. In human tumors, CPT1A expression and activity are decreased versus normal kidney; and poor patient outcome associates with lower expression of CPT1A in tumors in TCGA. Together, our studies identify HIF control of fatty acid metabolism as essential for ccRCC tumorigenesis.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma de Células Renais/metabolismo , Ácidos Graxos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Renais/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Carcinogênese , Carcinoma de Células Renais/genética , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Renais/genética , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitocôndrias/metabolismoRESUMO
Tandem MS "profiling" of acylcarnitines and amino acids was conceived as a first-tier screening method, and its application to expanded newborn screening has been enormously successful. However, unlike amino acid screening (which uses amino acid analysis as its second-tier validation of screening results), acylcarnitine "profiling" also assumed the role of second-tier validation, due to the lack of a generally accepted second-tier acylcarnitine determination method. In this report, we present results from the application of our validated UHPLC-MS/MS second-tier method for the quantification of total carnitine, free carnitine, butyrobetaine, and acylcarnitines to patient samples with known diagnoses: malonic acidemia, short-chain acyl-CoA dehydrogenase deficiency (SCADD) or isobutyryl-CoA dehydrogenase deficiency (IBD), 3-methyl-crotonyl carboxylase deficiency (3-MCC) or ß-ketothiolase deficiency (BKT), and methylmalonic acidemia (MMA). We demonstrate the assay's ability to separate constitutional isomers and diastereomeric acylcarnitines and generate values with a high level of accuracy and precision. These capabilities are unavailable when using tandem MS "profiles". We also show examples of research interest, where separation of acylcarnitine species and accurate and precise acylcarnitine quantification is necessary.
Assuntos
Acetil-CoA C-Aciltransferase/deficiência , Acil-CoA Desidrogenase/deficiência , Erros Inatos do Metabolismo dos Aminoácidos/diagnóstico , Carbono-Carbono Ligases/deficiência , Carnitina/análogos & derivados , Erros Inatos do Metabolismo Lipídico/diagnóstico , Distúrbios Congênitos do Ciclo da Ureia/diagnóstico , Acetil-CoA C-Aciltransferase/sangue , Acetil-CoA C-Aciltransferase/líquido cefalorraquidiano , Acetil-CoA C-Aciltransferase/urina , Acil-CoA Desidrogenase/sangue , Acil-CoA Desidrogenase/líquido cefalorraquidiano , Acil-CoA Desidrogenase/urina , Erros Inatos do Metabolismo dos Aminoácidos/sangue , Erros Inatos do Metabolismo dos Aminoácidos/líquido cefalorraquidiano , Erros Inatos do Metabolismo dos Aminoácidos/urina , Betaína/análogos & derivados , Betaína/sangue , Betaína/líquido cefalorraquidiano , Betaína/urina , Carbono-Carbono Ligases/sangue , Carbono-Carbono Ligases/líquido cefalorraquidiano , Carbono-Carbono Ligases/urina , Carnitina/sangue , Carnitina/líquido cefalorraquidiano , Carnitina/urina , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida de Alta Pressão/normas , Feminino , Humanos , Recém-Nascido , Isomerismo , Erros Inatos do Metabolismo Lipídico/sangue , Erros Inatos do Metabolismo Lipídico/líquido cefalorraquidiano , Erros Inatos do Metabolismo Lipídico/urina , Masculino , Triagem Neonatal , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem/normas , Distúrbios Congênitos do Ciclo da Ureia/sangue , Distúrbios Congênitos do Ciclo da Ureia/líquido cefalorraquidiano , Distúrbios Congênitos do Ciclo da Ureia/urinaRESUMO
A validated quantitative method for the determination of free and total carnitine, butyrobetaine, and acylcarnitines is presented. The versatile method has four components: (1) isolation using strong cation-exchange solid-phase extraction, (2) derivatization with pentafluorophenacyl trifluoromethanesulfonate, (3) sequential ion-exchange/reversed-phase (ultra) high-performance liquid chromatography [(U)HPLC] using a strong cation-exchange trap in series with a fused-core HPLC column, and (4) detection with electrospray ionization multiple reaction monitoring (MRM) mass spectrometry (MS). Standardized carnitine along with 65 synthesized, standardized acylcarnitines (including short-chain, medium-chain, long-chain, dicarboxylic, hydroxylated, and unsaturated acyl moieties) were used to construct multiple-point calibration curves, resulting in accurate and precise quantification. Separation of the 65 acylcarnitines was accomplished in a single chromatogram in as little as 14 min. Validation studies were performed showing a high level of accuracy, precision, and reproducibility. The method provides capabilities unavailable by tandem MS procedures, making it an ideal approach for confirmation of newborn screening results and for clinical and basic research projects, including treatment protocol studies, acylcarnitine biomarker studies, and metabolite studies using plasma, urine, tissue, or other sample matrixes.
Assuntos
Betaína/análogos & derivados , Carnitina/análogos & derivados , Carnitina/análise , Músculo Esquelético/química , Animais , Betaína/análise , Betaína/sangue , Betaína/urina , Carnitina/sangue , Carnitina/urina , Cromatografia Líquida de Alta Pressão , Diabetes Mellitus Experimental/diagnóstico , Humanos , Mesilatos/química , Ratos , Extração em Fase Sólida , Espectrometria de Massas por Ionização por Electrospray , Compostos de Trimetilsilil/químicaRESUMO
Previously we showed that in vivo treatment of elderly Fisher 344 rats with acetylcarnitine abolished the age-associated defect in respiratory chain complex III in interfibrillar mitochondria and improved the functional recovery of the ischemic/reperfused heart. Herein, we explored mitochondrial protein acetylation as a possible mechanism for acetylcarnitine's effect. In vivo treatment of elderly rats with acetylcarnitine restored cardiac acetylcarnitine content and increased mitochondrial protein lysine acetylation and increased the number of lysine-acetylated proteins in cardiac subsarcolemmal and interfibrillar mitochondria. Enzymes of the tricarboxylic acid cycle, mitochondrial ß-oxidation, and ATP synthase of the respiratory chain showed the greatest acetylation. Acetylation of isocitrate dehydrogenase, long-chain acyl-CoA dehydrogenase, complex V, and aspartate aminotransferase was accompanied by decreased catalytic activity. Several proteins were found to be acetylated only after treatment with acetylcarnitine, suggesting that exogenous acetylcarnitine served as the acetyl-donor. Two-dimensional fluorescence difference gel electrophoresis analysis revealed that acetylcarnitine treatment also induced changes in mitochondrial protein amount; a two-fold or greater increase/decrease in abundance was observed for thirty one proteins. Collectively, our data provide evidence for the first time that in the aged rat heart in vivo administration of acetylcarnitine provides acetyl groups for protein acetylation and affects the amount of mitochondrial proteins.
Assuntos
Acetilcarnitina/metabolismo , Envelhecimento/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/metabolismo , Miocárdio/metabolismo , Acetilação , Animais , Ratos , Ratos Endogâmicos F344RESUMO
Rat hearts were perfused with [1,2,3,4-(13)C4]palmitic acid (M+4), and the isotopic patterns of myocardial acylcarnitines and acyl-CoAs were analyzed using ultra-HPLC-MS/MS. The 91.2% (13)C enrichment in palmitoylcarnitine shows that little endogenous (M+0) palmitate contributed to its formation. The presence of M+2 myristoylcarnitine (95.7%) and M+2 acetylcarnitine (19.4%) is evidence for ß-oxidation of perfused M+4 palmitic acid. Identical enrichment data were obtained in the respective acyl-CoAs. The relative (13)C enrichment in M+4 (84.7%, 69.9%) and M+6 (16.2%, 17.8%) stearoyl- and arachidylcarnitine, respectively, clearly shows that the perfused palmitate is chain-elongated. The observed enrichment of (13)C in acetylcarnitine (19%), M+6 stearoylcarnitine (16.2%), and M+6 arachidylcarnitine (17.8%) suggests that the majority of two-carbon units for chain elongation are derived from ß-oxidation of [1,2,3,4-(13)C4]palmitic acid. These data are explained by conversion of the M+2 acetyl-CoA to M+2 malonyl-CoA, which serves as the acceptor for M+4 palmitoyl-CoA in chain elongation. Indeed, the (13)C enrichment in mitochondrial acetyl-CoA (18.9%) and malonyl-CoA (19.9%) are identical. No (13)C enrichment was found in acylcarnitine species with carbon chain lengths between 4 and 12, arguing against the simple reversal of fatty acid ß-oxidation. Furthermore, isolated, intact rat heart mitochondria 1) synthesize malonyl-CoA with simultaneous inhibition of carnitine palmitoyltransferase 1b and 2) catalyze the palmitoyl-CoA-dependent incorporation of (14)C from [2-(14)C]malonyl-CoA into lipid-soluble products. In conclusion, rat heart has the capability to chain-elongate fatty acids using mitochondria-derived two-carbon chain extenders. The data suggest that the chain elongation process is localized on the outer surface of the mitochondrial outer membrane.
Assuntos
Acetilcoenzima A/metabolismo , Inibidores Enzimáticos/farmacologia , Mitocôndrias Cardíacas/metabolismo , Miocárdio/metabolismo , Ácido Palmítico/metabolismo , Ácido Palmítico/farmacologia , Animais , Carnitina O-Palmitoiltransferase/metabolismo , Inibidores Enzimáticos/metabolismo , Malonil Coenzima A/metabolismo , Proteínas Musculares/metabolismo , Oxirredução , Palmitoil Coenzima A/metabolismo , Perfusão , Ratos , Ratos Endogâmicos F344RESUMO
Sterol regulatory element-binding protein-1 (SREBP-1) is a key transcription factor that regulates genes in the de novo lipogenesis and glycolysis pathways. The levels of SREBP-1 are significantly elevated in obese patients and in animal models of obesity and type 2 diabetes, and a vast number of studies have implicated this transcription factor as a contributor to hepatic lipid accumulation and insulin resistance. However, its role in regulating carbohydrate metabolism is poorly understood. Here we have addressed whether SREBP-1 is needed for regulating glucose homeostasis. Using RNAi and a new generation of adenoviral vector, we have silenced hepatic SREBP-1 in normal and obese mice. In normal animals, SREBP-1 deficiency increased Pck1 and reduced glycogen deposition during fed conditions, providing evidence that SREBP-1 is necessary to regulate carbohydrate metabolism during the fed state. Knocking SREBP-1 down in db/db mice resulted in a significant reduction in triglyceride accumulation, as anticipated. However, mice remained hyperglycemic, which was associated with up-regulation of gluconeogenesis gene expression as well as decreased glycolysis and glycogen synthesis gene expression. Furthermore, glycogen synthase activity and glycogen accumulation were significantly reduced. In conclusion, silencing both isoforms of SREBP-1 leads to significant changes in carbohydrate metabolism and does not improve insulin resistance despite reducing steatosis in an animal model of obesity and type 2 diabetes.
Assuntos
Regulação da Expressão Gênica/fisiologia , Gluconeogênese/fisiologia , Glicogênio/biossíntese , Fígado/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Animais , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Técnicas de Silenciamento de Genes , Glicogênio/genética , Masculino , Camundongos , Obesidade/genética , Obesidade/metabolismo , Obesidade/patologia , Proteína de Ligação a Elemento Regulador de Esterol 1/genéticaRESUMO
The mammalian heart, the body's largest energy consumer, has evolved robust mechanisms to tightly couple fuel supply with energy demand across a wide range of physiologic and pathophysiologic states, yet, when compared with other organs, relatively little is known about the molecular machinery that directly governs metabolic plasticity in the heart. Although previous studies have defined Kruppel-like factor 15 (KLF15) as a transcriptional repressor of pathologic cardiac hypertrophy, a direct role for the KLF family in cardiac metabolism has not been previously established. We show in human heart samples that KLF15 is induced after birth and reduced in heart failure, a myocardial expression pattern that parallels reliance on lipid oxidation. Isolated working heart studies and unbiased transcriptomic profiling in Klf15-deficient hearts demonstrate that KLF15 is an essential regulator of lipid flux and metabolic homeostasis in the adult myocardium. An important mechanism by which KLF15 regulates its direct transcriptional targets is via interaction with p300 and recruitment of this critical co-activator to promoters. This study establishes KLF15 as a key regulator of myocardial lipid utilization and is the first to implicate the KLF transcription factor family in cardiac metabolism.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Metabolismo dos Lipídeos , Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Cardiomegalia/genética , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Knockout , Proteínas Musculares/genética , Miocárdio/patologia , Proteínas Nucleares/genética , Oxirredução , Fatores de Transcrição/genéticaRESUMO
Clinical and animal studies have documented that hearts of the elderly are more susceptible to ischemia/reperfusion damage compared to young adults. Recently we found that aging-dependent increase in susceptibility of cardiomyocytes to apoptosis was attributable to decrease in cytosolic glutaredoxin 1 (Grx1) and concomitant decrease in NF-κB-mediated expression of anti-apoptotic proteins. Besides primary localization in the cytosol, Grx1 also exists in the mitochondrial intermembrane space (IMS). In contrast, Grx2 is confined to the mitochondrial matrix. Here we report that Grx1 is decreased by 50-60% in the IMS, but Grx2 is increased by 1.4-2.6 fold in the matrix of heart mitochondria from elderly rats. Determination of in situ activities of the Grx isozymes from both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria revealed that Grx1 was fully active in the IMS. However, Grx2 was mostly in an inactive form in the matrix, consistent with reversible sequestration of the active-site cysteines of two Grx2 molecules in complex with an iron-sulfur cluster. Our quantitative evaluations of the active/inactive ratio for Grx2 suggest that levels of dimeric Grx2 complex with iron-sulfur clusters are increased in SSM and IFM in the hearts of elderly rats. We found that the inactive Grx2 can be fully reactivated by sodium dithionite or exogenous superoxide production mediated by xanthine oxidase. However, treatment with rotenone, which generates intramitochondrial superoxide through inhibition of mitochondrial respiratory chain Complex I, did not lead to Grx2 activation. These findings suggest that insufficient ROS accumulates in the vicinity of dimeric Grx2 to activate it in situ.
Assuntos
Envelhecimento/metabolismo , Glutarredoxinas/metabolismo , Mitocôndrias Cardíacas/enzimologia , Animais , Mitocôndrias Cardíacas/metabolismo , Oxirredução , Ratos , Ratos Endogâmicos F344RESUMO
Mitochondrial reactive oxygen species (ROS) cause kidney damage in diabetes. We investigated the source and site of ROS production by kidney cortical tubule mitochondria in streptozotocin-induced type 1 diabetes in rats. In diabetic mitochondria, the increased amounts and activities of selective fatty acid oxidation enzymes is associated with increased oxidative phosphorylation and net ROS production with fatty acid substrates (by 40% and 30%, respectively), whereas pyruvate oxidation is decreased and pyruvate-supported ROS production is unchanged. Oxidation of substrates that donate electrons at specific sites in the electron transport chain (ETC) is unchanged. The increased maximal production of ROS with fatty acid oxidation is not affected by limiting the electron flow from complex I into complex III. The maximal capacity of the ubiquinol oxidation site in complex III in generating ROS does not differ between the control and diabetic mitochondria. In conclusion, the mitochondrial ETC is neither the target nor the site of ROS production in kidney tubule mitochondria in short-term diabetes. Mitochondrial fatty acid oxidation is the source of the increased net ROS production, and the site of electron leakage is located proximal to coenzyme Q at the electron transfer flavoprotein that shuttles electrons from acyl-CoA dehydrogenases to coenzyme Q.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Ácidos Graxos/metabolismo , Túbulos Renais Proximais/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Carnitina O-Palmitoiltransferase/metabolismo , Masculino , Mitocôndrias/metabolismo , Oxirredução , Ácido Pirúvico/metabolismo , Ratos , Ratos Wistar , Superóxido Dismutase/metabolismo , Ubiquinona/metabolismoRESUMO
The ability of skeletal muscle to enhance lipid utilization during exercise is a form of metabolic plasticity essential for survival. Conversely, metabolic inflexibility in muscle can cause organ dysfunction and disease. Although the transcription factor Kruppel-like factor 15 (KLF15) is an important regulator of glucose and amino acid metabolism, its endogenous role in lipid homeostasis and muscle physiology is unknown. Here we demonstrate that KLF15 is essential for skeletal muscle lipid utilization and physiologic performance. KLF15 directly regulates a broad transcriptional program spanning all major segments of the lipid-flux pathway in muscle. Consequently, Klf15-deficient mice have abnormal lipid and energy flux, excessive reliance on carbohydrate fuels, exaggerated muscle fatigue, and impaired endurance exercise capacity. Elucidation of this heretofore unrecognized role for KLF15 now implicates this factor as a central component of the transcriptional circuitry that coordinates physiologic flux of all three basic cellular nutrients: glucose, amino acids, and lipids.
Assuntos
Exercício Físico , Fatores de Transcrição Kruppel-Like/fisiologia , Metabolismo dos Lipídeos , Músculo Esquelético/metabolismo , Proteínas Nucleares/fisiologia , Aminoácidos/metabolismo , Glucose/metabolismo , Homeostase , HumanosRESUMO
Voltage-dependent anion channels are abundant mitochondrial outer membrane proteins expressed in three isoforms, VDAC1-3, and are considered as "mitochondrial gatekeepers". Most tissues express all three isoforms. The functions of VDACs are several-fold, ranging from metabolite and energy exchange to apoptosis. Some of these functions depend on or are affected by interaction with other proteins in the cytosol and intermembrane space. Furthermore, the function of VDACs, as well as their interaction with other proteins, is affected by posttranslational modification, mainly phosphorylation. This review summarizes recent findings on posttranslational modification of VDACs and discusses the physiological outcome of these modifications. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.
Assuntos
Processamento de Proteína Pós-Traducional , Proteômica , Canais de Ânion Dependentes de Voltagem/metabolismo , Humanos , Espectrometria de Massas , Isoformas de Proteínas/metabolismo , Canais de Ânion Dependentes de Voltagem/químicaRESUMO
CPT1a (carnitine palmitoyltransferase 1a) in the liver mitochondrial outer membrane (MOM) catalyzes the primary regulated step in overall mitochondrial fatty acid oxidation. It has been suggested that the fundamental unit of CPT1a exists as a trimer, which, under native conditions, could form a dimer of the trimers, creating a hexamer channel for acylcarnitine translocation. To examine the state of CPT1a in the MOM, we employed a combined approach of sizing by mass and isolation using an immunological method. Blue native electrophoresis followed by detection with immunoblotting and mass spectrometry identified large molecular mass complexes that contained not only CPT1a but also long chain acyl-CoA synthetase (ACSL) and the voltage-dependent anion channel (VDAC). Immunoprecipitation with antisera against the proteins revealed a strong interaction between the three proteins. Immobilized CPT1a-specific antibodies immunocaptured not only CPT1a but also ACSL and VDAC, further strengthening findings with blue native electrophoresis and immunoprecipitation. This study shows strong protein-protein interaction between CPT1a, ACSL, and VDAC. We propose that this complex transfers activated fatty acids through the MOM.
Assuntos
Carnitina O-Palmitoiltransferase/metabolismo , Ácidos Graxos/metabolismo , Mitocôndrias/enzimologia , Membranas Mitocondriais/metabolismo , Animais , Transporte Biológico , Carnitina O-Palmitoiltransferase/química , Carnitina O-Palmitoiltransferase/isolamento & purificação , Coenzima A Ligases/química , Coenzima A Ligases/isolamento & purificação , Coenzima A Ligases/metabolismo , Eletroforese , Imunoprecipitação , Fígado/citologia , Masculino , Mitocôndrias/metabolismo , Membranas Mitocondriais/enzimologia , Peso Molecular , Multimerização Proteica , Estrutura Quaternária de Proteína , Ratos , Ratos Sprague-Dawley , Canais de Ânion Dependentes de Voltagem/química , Canais de Ânion Dependentes de Voltagem/isolamento & purificação , Canais de Ânion Dependentes de Voltagem/metabolismoRESUMO
The mitochondrial outer membrane surrounds the entire organelle. It is composed of a phospholipid bilayer with proteins either embedded into or anchored to the bilayer and mediates the interactions between mitochondria and the rest of the cell. Most of the proteins present in the mitochondrial outer membrane are highly hydrophobic with one or more transmembrane segments. These proteins in conjunction with proteins localized in the inner membrane catalyse energy exchange reactions, the flux of small molecules such as ions, the activation and uptake of long chain fatty acids, import of proteins into the mitochondria, and elimination of biogenic amines among others. In addition, some outer membrane proteins serve as docking sites for non-resident enzymes such as hexokinase and other kinases of signal transduction. All these processes require an intact outer membrane and are highly regulated. One level of regulation with physiological/pathophysiological relevance involves post-translational modification of outer membrane proteins, either by phosphorylation, acetylation or other type of reversible covalent modification. Post-translational modification such as nitration and carbonylation becomes significant under disease states that are associated with increased oxidative stress, i.e. inflammation and ischemia. This review examines the different post-translational modifications of mitochondrial outer membrane proteins and discusses the physiological relevance of these modifications.
Assuntos
Proteínas de Membrana/metabolismo , Membranas Mitocondriais/metabolismo , Processamento de Proteína Pós-Traducional , Acetilação , Carnitina O-Palmitoiltransferase/metabolismo , Coenzima A Ligases/metabolismo , Humanos , Membranas Mitocondriais/enzimologia , Fosforilação , Ligação ProteicaRESUMO
In recent years, a wide variety of proteomic approaches using gel electrophoresis and mass spectrometry has been developed to detect post-translational modifications. Mitochondria are often a focus of these studies due to their important role in cellular function. Many of their crucial transport and oxidative-phosphorylation functions are performed by proteins residing in the inner and outer membranes of the mitochondria. Although proteomic technologies have greatly enhanced our understanding of regulation in cellular processes, analysis of membrane proteins has lagged behind that of soluble proteins. Herein, we present techniques to facilitate the detection of post-translational modifications of mitochondrial membrane proteins including the isolation of resident membranes as well as electrophoretic and immunological-based methods for identification of post-translational modifications.
Assuntos
Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/análise , Proteínas Mitocondriais/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Sequência de Aminoácidos , Animais , Carnitina O-Palmitoiltransferase/análise , Carnitina O-Palmitoiltransferase/isolamento & purificação , Carnitina O-Palmitoiltransferase/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Espectrometria de Massas , Membranas Mitocondriais/química , Proteínas Mitocondriais/isolamento & purificação , Dados de Sequência MolecularRESUMO
The carnitine palmitoyltransferase-I (CPT-I) enzymes catalyze the regulated step in overall mitochondrial fatty acid oxidation. The liver and muscle isoforms are expressed in liver and skeletal muscle respectively with the isoforms exhibiting different kinetic properties and apparent molecular weight masses. In contrast, the heart expresses both isoforms at the mRNA level. However, for the expression of the liver isoform at the protein level only indirect evidence is available, such as tagging with radiolabeled CPT-I inhibitors followed by SDS-PAGE separation and kinetic analysis using inhibitors. The importance of fatty acid oxidation in the heart and the potential regulation via the liver isoform of CPT-I demands proof of the liver isoform in the heart. Using a proteomic approach in the present study we demonstrate that rat heart mitochondria (a) contain both the muscle and liver isoforms; (b) both proteins retain their C- and N-termini; (c) the N-terminal alanine residues are acetylated; (d) and in rat heart mitochondria the liver isoform is phosphorylated on tyrosine 281. By providing amino acid sequence information this is the first unequivocal demonstration that the liver isoform of CPT-I is expressed at the protein level in adult rat heart mitochondria and that the apparent smaller molecular size of the muscle isoform is not due to proteolytic truncation.
Assuntos
Carnitina O-Palmitoiltransferase/metabolismo , Mitocôndrias Cardíacas/enzimologia , Sequência de Aminoácidos , Animais , Isoenzimas/metabolismo , Masculino , Mitocôndrias Hepáticas/enzimologia , Dados de Sequência Molecular , Peso Molecular , Músculos/enzimologia , Fosforilação , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
For the proteomic study of mitochondrial membranes, documented high quality mitochondrial preparations are a necessity to ensure proper localization. Despite the state-of-the-art technologies currently in use, there is no single technique that can be used for all studies of mitochondrial membrane proteins. Herein, we use examples to highlight solubilization techniques, different chromatographic methods, and developments in gel electrophoresis for proteomic analysis of mitochondrial membrane proteins. Blue-native gel electrophoresis has been successful not only for dissection of the inner membrane oxidative phosphorylation system, but also for the components of the outer membrane such as those involved in protein import. Identification of PTMs such as phosphorylation, acetylation, and nitration of mitochondrial membrane proteins has been greatly improved by the use of affinity techniques. However, understanding of the biological effect of these modifications is an area for further exploration. The rapid development of proteomic methods for both identification and quantitation, especially for modifications, will greatly impact the understanding of the mitochondrial membrane proteome.
Assuntos
Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Proteômica/métodos , Animais , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Humanos , Proteínas de Membrana/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
AIMS: Mitochondrial dysfunction is a major factor in heart failure (HF). A pronounced variability of mitochondrial electron transport chain (ETC) defects is reported to occur in severe acquired cardiomyopathies without a consistent trend for depressed activity or expression. The aim of this study was to define the defect in the integrative function of cardiac mitochondria in coronary microembolization-induced HF. METHODS AND RESULTS: Studies were performed in the canine coronary microembolization-induced HF model of moderate severity. Oxidative phosphorylation was assessed as the integrative function of mitochondria, using a comprehensive variety of substrates in order to investigate mitochondrial membrane transport, dehydrogenase activity and electron-transport coupled to ATP synthesis. The supramolecular organization of the mitochondrial ETC also was investigated by native gel electrophoresis. We found a dramatic decrease in ADP-stimulated respiration that was not relieved by an uncoupler. Moreover, the ADP/O ratio was normal, indicating no defect in the phosphorylation apparatus. The data point to a defect in oxidative phosphorylation within the ETC. However, the individual activities of ETC complexes were normal. The amount of the supercomplex consisting of complex I/complex III dimer/complex IV, the major form of respirasome considered essential for oxidative phosphorylation, was decreased. CONCLUSIONS: We propose that the mitochondrial defect lies in the supermolecular assembly rather than in the individual components of the ETC.
Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Insuficiência Cardíaca/metabolismo , Mitocôndrias Cardíacas/metabolismo , Fosforilação Oxidativa , Trifosfato de Adenosina/biossíntese , Animais , Respiração Celular , Cães , Transporte de Elétrons , Hemodinâmica , Proteínas de Membrana Transportadoras/metabolismo , Miopatias Mitocondriais/metabolismo , Oxirredutases/metabolismoRESUMO
BACKGROUND: Analysis of carnitine and acylcarnitines by tandem mass spectrometry (MS/MS) has limitations. First, preparation of butyl esters partially hydrolyzes acylcarnitines. Second, isobaric nonacylcarnitine compounds yield false-positive results in acylcarnitine tests. Third, acylcarnitine constitutional isomers cannot be distinguished. METHODS: Carnitine and acylcarnitines were isolated by ion-exchange solid-phase extraction, derivatized with pentafluorophenacyl trifluoromethanesulfonate, separated by HPLC, and detected with an ion trap mass spectrometer. Carnitine was quantified with d(3)-carnitine as the internal standard. Acylcarnitines were quantified with 42 synthesized calibrators. The internal standards used were d(6)-acetyl-, d(3)-propionyl-, undecanoyl-, undecanedioyl-, and heptadecanoylcarnitine. RESULTS: Example recoveries [mean (SD)] were 69.4% (3.9%) for total carnitine, 83.1% (5.9%) for free carnitine, 102.2% (9.8%) for acetylcarnitine, and 107.2% (8.9%) for palmitoylcarnitine. Example imprecision results [mean (SD)] within runs (n = 6) and between runs (n = 18) were, respectively: total carnitine, 58.0 (0.9) and 57.4 (1.7) micromol/L; free carnitine, 44.6 (1.5) and 44.3 (1.2) micromol/L; acetylcarnitine, 7.74 (0.51) and 7.85 (0.69) micromol/L; and palmitoylcarnitine, 0.12 (0.01) and 0.11 (0.02) micromol/L. Standard-addition slopes and linear regression coefficients were 1.00 and 0.9998, respectively, for total carnitine added to plasma, 0.99 and 0.9997 for free carnitine added to plasma, 1.04 and 0.9972 for octanoylcarnitine added to skeletal muscle, and 1.05 and 0.9913 for palmitoylcarnitine added to skeletal muscle. Reference intervals for plasma, urine, and skeletal muscle are provided. CONCLUSIONS: This method for analysis of carnitine and acylcarnitines overcomes the observed limitations of MS/MS methods.
Assuntos
Carnitina/análise , Carnitina/química , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Acetilação , Calibragem , Carnitina/metabolismo , Humanos , Estrutura MolecularRESUMO
Hepatic mitochondrial fatty acid oxidation and ketogenesis increase during starvation. Carnitine palmitoyltransferase I (CPT-I) catalyses the rate-controlling step in the overall pathway and retains its control over beta-oxidation under fed, starved and diabetic conditions. To determine the factors contributing to the reported several-fold increase in fatty acid oxidation in perfused livers, we measured the V(max) and K(m) values for palmitoyl-CoA and carnitine, the K(i) (and IC(50)) values for malonyl-CoA in isolated liver mitochondria as well as the hepatic malonyl-CoA and carnitine contents in control and 48 h starved rats. Since CPT-I is localized in the mitochondrial outer membrane and in contact sites, the kinetic properties of CPT-I also was determined in these submitochondrial structures. After 48 h starvation, there is: (a) a significant increase in K(i) and decrease in hepatic malonyl-CoA content; (b) a decreased K(m) for palmitoyl-CoA; and (c) increased catalytic activity (V(max)) and CPT-I protein abundance that is significantly greater in contact sites compared with outer membranes. Based on these changes the estimated increase in mitochondrial fatty acid oxidation is significantly less than that observed in perfused liver. This suggests that CPT-I is regulated in vivo by additional mechanism(s) lost during mitochondrial isolation or/and that mitochondrial oxidation of peroxisomal beta-oxidation products contribute to the increased ketogenesis by bypassing CPT-I. Furthermore, the greater increase in CPT-I protein in contact sites as compared to outer membranes emphasizes the significance of contact sites in hepatic fatty acid oxidation.
Assuntos
Carnitina O-Palmitoiltransferase/metabolismo , Carnitina/metabolismo , Fígado/metabolismo , Malonil Coenzima A/metabolismo , Mitocôndrias Hepáticas/enzimologia , Inanição/metabolismo , Animais , Western Blotting , Peso Corporal , Eletroforese em Gel de Poliacrilamida , Masculino , Tamanho do Órgão , Ratos , Ratos Sprague-DawleyRESUMO
A novel procedure for the quantitative isolation and purification of acyl-coenzyme A esters is presented. The procedure involves two steps: (1) tissue extraction using acetonitrile/2-propanol (3+1, v+v) followed by 0.1M potassium phosphate, pH 6.7, and (2) purification using 2-(2-pyridyl)ethyl-functionalized silica gel. Recoveries determined by adding radiolabeled acetyl-, malonyl-, octanoyl-, oleoyl-, palmitoyl-, or arachidonyl-coenzyme A to powdered rat liver varied 93-104% for tissue extraction and 83-90% for solid-phase extraction. The procedure described allows for isolation and purification, with high recoveries, of acyl-coenzyme A esters differing widely in chain length and saturation.