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1.
J Inherit Metab Dis ; 47(2): 280-288, 2024 03.
Artigo em Inglês | MEDLINE | ID: mdl-38200664

RESUMO

Glyoxylate is a key metabolite generated from various precursor substrates in different subcellular compartments including mitochondria, peroxisomes, and the cytosol. The fact that glyoxylate is a good substrate for the ubiquitously expressed enzyme lactate dehydrogenase (LDH) requires the presence of efficient glyoxylate detoxification systems to avoid the formation of oxalate. Furthermore, this detoxification needs to be compartment-specific since LDH is actively present in multiple subcellular compartments including peroxisomes, mitochondria, and the cytosol. Whereas the identity of these protection systems has been established for both peroxisomes and the cytosol as concluded from the deficiency of alanine glyoxylate aminotransferase (AGT) in primary hyperoxaluria type 1 (PH1) and glyoxylate reductase (GR) in PH2, the glyoxylate protection system in mitochondria has remained less well defined. In this manuscript, we show that the enzyme glyoxylate reductase has a bimodal distribution in human embryonic kidney (HEK293), hepatocellular carcinoma (HepG2), and cervical carcinoma (HeLa) cells and more importantly, in human liver, and is actively present in both the mitochondrial and cytosolic compartments. We conclude that the metabolism of glyoxylate in humans requires the complicated interaction between different subcellular compartments within the cell and discuss the implications for the different primary hyperoxalurias.


Assuntos
Oxirredutases do Álcool , Mitocôndrias Hepáticas , Transaminases , Humanos , Mitocôndrias Hepáticas/metabolismo , Células HEK293 , Oxalatos/metabolismo , Fígado/metabolismo , Glioxilatos/metabolismo
2.
Genet Med ; 23(4): 740-750, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33239752

RESUMO

PURPOSE: In this study we investigate the disease etiology in 12 patients with de novo variants in FAR1 all resulting in an amino acid change at position 480 (p.Arg480Cys/His/Leu). METHODS: Following next-generation sequencing and clinical phenotyping, functional characterization was performed in patients' fibroblasts using FAR1 enzyme analysis, FAR1 immunoblotting/immunofluorescence, and lipidomics. RESULTS: All patients had spastic paraparesis and bilateral congenital/juvenile cataracts, in most combined with speech and gross motor developmental delay and truncal hypotonia. FAR1 deficiency caused by biallelic variants results in defective ether lipid synthesis and plasmalogen deficiency. In contrast, patients' fibroblasts with the de novo FAR1 variants showed elevated plasmalogen levels. Further functional studies in fibroblasts showed that these variants cause a disruption of the plasmalogen-dependent feedback regulation of FAR1 protein levels leading to uncontrolled ether lipid production. CONCLUSION: Heterozygous de novo variants affecting the Arg480 residue of FAR1 lead to an autosomal dominant disorder with a different disease mechanism than that of recessive FAR1 deficiency and a diametrically opposed biochemical phenotype. Our findings show that for patients with spastic paraparesis and bilateral cataracts, FAR1 should be considered as a candidate gene and added to gene panels for hereditary spastic paraplegia, cerebral palsy, and juvenile cataracts.


Assuntos
Aldeído Oxirredutases/genética , Éteres , Lipídeos , Paraplegia Espástica Hereditária/genética , Humanos , Fenótipo
3.
J Inherit Metab Dis ; 44(2): 438-449, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32740932

RESUMO

Post-translational protein modifications derived from metabolic intermediates, such as acyl-CoAs, have been shown to regulate mitochondrial function. Patients with a genetic defect in the propionyl-CoA carboxylase (PCC) gene clinically present symptoms related to mitochondrial disorders and are characterised by decreased mitochondrial respiration. Since propionyl-CoA accumulates in PCC deficient patients and protein propionylation can be driven by the level of propionyl-CoA, we hypothesised that protein propionylation could play a role in the pathology of the disease. Indeed, we identified increased protein propionylation due to pathologic propionyl-CoA accumulation in patient-derived fibroblasts and this was accompanied by defective mitochondrial respiration, as was shown by a decrease in complex I-driven respiration. To mimic pathological protein propionylation levels, we exposed cultured fibroblasts, Fao liver cells and C2C12 muscle myotubes to propionate levels that are typically found in these patients. This induced a global increase in protein propionylation and histone protein propionylation and was also accompanied by a decrease in mitochondrial respiration in liver and fibroblasts. However, in C2C12 myotubes propionate exposure did not decrease mitochondrial respiration, possibly due to differences in propionyl-CoA metabolism as compared to the liver. Therefore, protein propionylation could contribute to the pathology in these patients, especially in the liver, and could therefore be an interesting target to pursue in the treatment of this metabolic disease.


Assuntos
Fibroblastos/metabolismo , Metilmalonil-CoA Descarboxilase/genética , Mitocôndrias/genética , Fibras Musculares Esqueléticas/metabolismo , Acidemia Propiônica/genética , Humanos , Fígado/metabolismo , Proteínas de Membrana , Mitocôndrias/enzimologia , Propionatos/metabolismo , Acidemia Propiônica/enzimologia , Processamento de Proteína Pós-Traducional/genética
4.
Hum Mutat ; 40(10): 1899-1904, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31187905

RESUMO

Primary carnitine deficiency is caused by a defect in the active cellular uptake of carnitine by Na+ -dependent organic cation transporter novel 2 (OCTN2). Genetic diagnostic yield for this metabolic disorder has been relatively low, suggesting that disease-causing variants are missed. We Sanger sequenced the 5' untranslated region (UTR) of SLC22A5 in individuals with possible primary carnitine deficiency in whom no or only one mutant allele had been found. We identified a novel 5'-UTR c.-149G>A variant which we characterized by expression studies with reporter constructs in HeLa cells and by carnitine-transport measurements in fibroblasts using a newly developed sensitive assay based on tandem mass spectrometry. This variant, which we identified in 57 of 236 individuals of our cohort, introduces a functional upstream out-of-frame translation initiation codon. We show that the codon suppresses translation from the wild-type ATG of SLC22A5, resulting in reduced OCTN2 protein levels and concomitantly lower transport activity. With an allele frequency of 24.2% the c.-149G>A variant is the most frequent cause of primary carnitine deficiency in our cohort and may explain other reported cases with an incomplete genetic diagnosis. Individuals carrying this variant should be clinically re-evaluated and monitored to determine if this variant has clinical consequences.


Assuntos
Regiões 5' não Traduzidas , Cardiomiopatias/genética , Carnitina/deficiência , Códon de Iniciação , Predisposição Genética para Doença , Hiperamonemia/genética , Doenças Musculares/genética , Mutação , Membro 5 da Família 22 de Carreadores de Soluto/genética , Alelos , Sequência de Aminoácidos , Sequência de Bases , Transporte Biológico , Cardiomiopatias/diagnóstico , Cardiomiopatias/metabolismo , Carnitina/genética , Carnitina/metabolismo , Linhagem Celular , Frequência do Gene , Genes Reporter , Estudos de Associação Genética , Humanos , Hiperamonemia/diagnóstico , Hiperamonemia/metabolismo , Doenças Musculares/diagnóstico , Doenças Musculares/metabolismo , Membro 5 da Família 22 de Carreadores de Soluto/metabolismo
5.
Ann Neurol ; 82(5): 686-696, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29023963

RESUMO

OBJECTIVE: Mucopolysaccharidosis IIIA or Sanfilippo disease type A is a progressive neurodegenerative disorder presenting in early childhood, caused by an inherited deficiency of the lysosomal hydrolase sulfamidase. New missense mutations, for which genotype-phenotype correlations are currently unknown, are frequently reported, hampering early prediction of phenotypic severity and efficacy assessment of new disease-modifying treatments. We aimed to design a method to determine phenotypic severity early in the disease course. METHODS: Fifty-three patients were included for whom skin fibroblasts and data on disease course and mutation analysis were available. Patients were phenotypically characterized on clinical data as rapidly progressing or slowly progressing. Sulfamidase activity was measured in fibroblasts cultured at 37 °C and at 30 °C. RESULTS: Sulfamidase activity in fibroblasts from patients homozygous or compound heterozygous for a combination of known severe mutations remained below the limit of quantification under both culture conditions. In contrast, sulfamidase activity in fibroblasts from patients homozygous or compound heterozygous for a known mild mutation increased above the limit of quantification when cultured at 30 °C. With division on the basis of the patients' phenotype, fibroblasts from slowly progressing patients could be separated from rapidly progressing patients by increase in sulfamidase activity when cultured at 30 °C (p < 0.001, sensitivity = 96%, specificity = 93%). INTERPRETATION: Phenotypic severity strongly correlates with the potential to increase sulfamidase activity in fibroblasts cultured at 30 °C, allowing reliable distinction between patients with rapidly progressing or slowly progressing phenotypes. This method may provide an essential tool for assessment of treatment effects and for health care and life planning decisions. Ann Neurol 2017;82:686-696.


Assuntos
Fibroblastos/metabolismo , Hidrolases/metabolismo , Mucopolissacaridose III/diagnóstico , Mucopolissacaridose III/enzimologia , Adolescente , Adulto , Técnicas de Cultura de Células , Células Cultivadas , Criança , Pré-Escolar , Progressão da Doença , Feminino , Humanos , Limite de Detecção , Masculino , Fenótipo , Valor Preditivo dos Testes , Temperatura , Adulto Jovem
6.
Mol Cell Proteomics ; 14(11): 3056-71, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26320211

RESUMO

The protein substrates of sirtuin 5-regulated lysine malonylation (Kmal) remain unknown, hindering its functional analysis. In this study, we carried out proteomic screening, which identified 4042 Kmal sites on 1426 proteins in mouse liver and 4943 Kmal sites on 1822 proteins in human fibroblasts. Increased malonyl-CoA levels in malonyl-CoA decarboxylase (MCD)-deficient cells induces Kmal levels in substrate proteins. We identified 461 Kmal sites showing more than a 2-fold increase in response to MCD deficiency as well as 1452 Kmal sites detected only in MCD-/- fibroblast but not MCD+/+ cells, suggesting a pathogenic role of Kmal in MCD deficiency. Cells with increased lysine malonylation displayed impaired mitochondrial function and fatty acid oxidation, suggesting that lysine malonylation plays a role in pathophysiology of malonic aciduria. Our study establishes an association between Kmal and a genetic disease and offers a rich resource for elucidating the contribution of the Kmal pathway and malonyl-CoA to cellular physiology and human diseases.


Assuntos
Carboxiliases/deficiência , Fígado/metabolismo , Lisina/metabolismo , Malonatos/metabolismo , Erros Inatos do Metabolismo/metabolismo , Mitocôndrias/metabolismo , Animais , Carboxiliases/genética , Carboxiliases/metabolismo , Linhagem Celular , Ácidos Graxos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Fígado/patologia , Masculino , Malonil Coenzima A/genética , Malonil Coenzima A/metabolismo , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/patologia , Ácido Metilmalônico/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/patologia , Modelos Moleculares , Oxirredução , Sirtuínas/deficiência , Sirtuínas/genética
7.
Hum Mol Genet ; 23(5): 1311-9, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24158852

RESUMO

Oxidative phosphorylation and fatty acid oxidation are two major metabolic pathways in mitochondria. Acyl-CoA dehydrogenase 9 (ACAD9), an enzyme assumed to play a role in fatty acid oxidation, was recently identified as a factor involved in complex I biogenesis. Here we further investigated the role of ACAD9's enzymatic activity in fatty acid oxidation and complex I biogenesis. We provide evidence indicating that ACAD9 displays enzyme activity in vivo. Knockdown experiments in very-long-chain acyl-CoA dehydrogenase (VLCAD)-deficient fibroblasts revealed that ACAD9 is responsible for the production of C14:1-carnitine from oleate and C12-carnitine from palmitate. These results explain the origin of these obscure acylcarnitines that are used to diagnose VLCAD deficiency in humans. Knockdown of ACAD9 in control fibroblasts did not reveal changes in the acylcarnitine profiles upon fatty acid loading. Next, we investigated whether catalytic activity of ACAD9 was necessary for complex I biogenesis. Catalytically inactive ACAD9 gave partial-to-complete rescue of complex I biogenesis in ACAD9-deficient cells and was incorporated in high-molecular-weight assembly intermediates. Our results underscore the importance of the ACAD9 protein in complex I assembly and suggest that the enzymatic activity is a rudiment of the duplication event.


Assuntos
Acil-CoA Desidrogenases/metabolismo , Ácidos Graxos/metabolismo , Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Acil-CoA Desidrogenase de Cadeia Longa/metabolismo , Acil-CoA Desidrogenases/química , Acil-CoA Desidrogenases/deficiência , Acil-CoA Desidrogenases/genética , Carnitina/biossíntese , Catálise , Linhagem Celular , Síndrome Congênita de Insuficiência da Medula Óssea , Complexo I de Transporte de Elétrons/deficiência , Ativação Enzimática , Humanos , Erros Inatos do Metabolismo Lipídico/metabolismo , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Modelos Moleculares , Peso Molecular , Doenças Musculares/metabolismo , Mutação , Oxirredução , Fosforilação Oxidativa , Conformação Proteica
8.
Hum Mol Genet ; 23(13): 3513-22, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24516071

RESUMO

Mitochondria integrate metabolic networks for maintaining bioenergetic requirements. Deregulation of mitochondrial metabolic networks can lead to mitochondrial dysfunction, which is a common hallmark of many diseases. Reversible post-translational protein acetylation modifications are emerging as critical regulators of mitochondrial function and form a direct link between metabolism and protein function, via the metabolic intermediate acetyl-CoA. Sirtuins catalyze protein deacetylation, but how mitochondrial acetylation is determined is unclear. We report here a mechanism that explains mitochondrial protein acetylation dynamics in vivo. Food withdrawal in mice induces a rapid increase in hepatic protein acetylation. Furthermore, using a novel LC-MS/MS method, we were able to quantify protein acetylation in human fibroblasts. We demonstrate that inducing fatty acid oxidation in fibroblasts increases protein acetylation. Furthermore, we show by using radioactively labeled palmitate that fatty acids are a direct source for mitochondrial protein acetylation. Intriguingly, in a mouse model that resembles human very-long chain acyl-CoA dehydrogenase (VLCAD) deficiency, we demonstrate that upon food-withdrawal, hepatic protein hyperacetylation is absent. This indicates that functional fatty acid oxidation is necessary for protein acetylation to occur in the liver upon food withdrawal. Furthermore, we now demonstrate that protein acetylation is abundant in human liver peroxisomes, an organelle where acetyl-CoA is solely generated by fatty acid oxidation. Our findings provide a mechanism for metabolic control of protein acetylation, which provides insight into the pathophysiogical role of protein acetylation dynamics in fatty acid oxidation disorders and other metabolic diseases associated with mitochondrial dysfunction.


Assuntos
Acetilcoenzima A/metabolismo , Ácidos Graxos/metabolismo , Acetilação , Animais , Western Blotting , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Fibroblastos/metabolismo , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução , Peroxissomos/metabolismo , Espectrometria de Massas em Tandem
9.
Hum Mol Genet ; 23(18): 5009-16, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-24847004

RESUMO

Dienoyl-CoA reductase (DECR) deficiency with hyperlysinemia is a rare disorder affecting the metabolism of polyunsaturated fatty acids and lysine. The molecular basis of this condition is currently unknown. We describe a new case with failure to thrive, developmental delay, lactic acidosis and severe encephalopathy suggestive of a mitochondrial disorder. Exome sequencing revealed a causal mutation in NADK2. NADK2 encodes the mitochondrial NAD kinase, which is crucial for NADP biosynthesis evidenced by decreased mitochondrial NADP(H) levels in patient fibroblasts. DECR and also the first step in lysine degradation are performed by NADP-dependent oxidoreductases explaining their in vivo deficiency. DECR activity was also deficient in lysates of patient fibroblasts and could only be rescued by transfecting patient cells with functional NADK2. Thus NADPH is not only crucial as a cosubstrate, but can also act as a molecular chaperone that activates and stabilizes enzymes. In addition to polyunsaturated fatty acid oxidation and lysine degradation, NADPH also plays a role in various other mitochondrial processes. We found decreased oxygen consumption and increased extracellular acidification in patient fibroblasts, which may explain why the disease course is consistent with clinical criteria for a mitochondrial disorder. We conclude that DECR deficiency with hyperlysinemia is caused by mitochondrial NADP(H) deficiency due to a mutation in NADK2.


Assuntos
Hiperlisinemias/genética , Proteínas Mitocondriais/genética , NADP/deficiência , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/deficiência , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fibroblastos/metabolismo , Humanos , Hiperlisinemias/fisiopatologia , Mutação , Análise de Sequência de DNA , Estresse Fisiológico
11.
Hum Mol Genet ; 22(25): 5249-61, 2013 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-23933733

RESUMO

The importance of mitochondrial fatty acid ß-oxidation (FAO) as a glucose-sparing process is illustrated by patients with inherited defects in FAO, who may present with life-threatening fasting-induced hypoketotic hypoglycemia. It is unknown why peripheral glucose demand outpaces hepatic gluconeogenesis in these patients. In this study, we have systematically addressed the fasting response in long-chain acyl-CoA dehydrogenase-deficient (LCAD KO) mice. We demonstrate that the fasting-induced hypoglycemia in LCAD KO mice was initiated by an increased glucose requirement in peripheral tissues, leading to rapid hepatic glycogen depletion. Gluconeogenesis did not compensate for the increased glucose demand, which was not due to insufficient hepatic glucogenic capacity but rather caused by a shortage in the supply of glucogenic precursors. This shortage in supply was explained by a suppressed glucose-alanine cycle, decreased branched-chain amino acid metabolism and ultimately impaired protein mobilization. We conclude that during fasting, FAO not only serves to spare glucose but is also indispensable for amino acid metabolism, which is essential for the maintenance of adequate glucose production.


Assuntos
Gluconeogênese/genética , Glucose/metabolismo , Hipoglicemia/metabolismo , Oxirredução , Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Acil-CoA Desidrogenase de Cadeia Longa/genética , Acil-CoA Desidrogenase de Cadeia Longa/metabolismo , Aminoácidos/metabolismo , Animais , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Humanos , Hipoglicemia/genética , Hipoglicemia/patologia , Erros Inatos do Metabolismo Lipídico/metabolismo , Glicogênio Hepático/genética , Glicogênio Hepático/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/patologia
12.
J Inherit Metab Dis ; 38(5): 873-9, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25860818

RESUMO

Alpha-aminoadipic and alpha-ketoadipic aciduria is an autosomal recessive inborn error of lysine, hydroxylysine, and tryptophan degradation. To date, DHTKD1 mutations have been reported in two alpha-aminoadipic and alpha-ketoadipic aciduria patients. We have now sequenced DHTKD1 in nine patients diagnosed with alpha-aminoadipic and alpha-ketoadipic aciduria as well as one patient with isolated alpha-aminoadipic aciduria, and identified causal mutations in eight. We report nine novel mutations, including three missense mutations, two nonsense mutations, two splice donor mutations, one duplication, and one deletion and insertion. Two missense mutations, one of which was reported before, were observed in the majority of cases. The clinical presentation of this group of patients was inhomogeneous. Our results confirm that alpha-aminoadipic and alpha-ketoadipic aciduria is caused by mutations in DHTKD1, and further establish that DHTKD1 encodes the E1 subunit of the alpha-ketoadipic acid dehydrogenase complex.


Assuntos
Ácido 2-Aminoadípico/metabolismo , Adipatos/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/genética , Cetona Oxirredutases/genética , Ácido 2-Aminoadípico/urina , Adipatos/urina , Adolescente , Adulto , Erros Inatos do Metabolismo dos Aminoácidos/diagnóstico , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Pré-Escolar , Feminino , Humanos , Recém-Nascido , Complexo Cetoglutarato Desidrogenase , Cetona Oxirredutases/deficiência , Cetona Oxirredutases/metabolismo , Masculino , Adulto Jovem
13.
Biochim Biophys Acta ; 1831(9): 1467-74, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23850792

RESUMO

Fatty acid ß-oxidation may occur in both mitochondria and peroxisomes. While peroxisomes oxidize specific carboxylic acids such as very long-chain fatty acids, branched-chain fatty acids, bile acids, and fatty dicarboxylic acids, mitochondria oxidize long-, medium-, and short-chain fatty acids. Oxidation of long-chain substrates requires the carnitine shuttle for mitochondrial access but medium-chain fatty acid oxidation is generally considered carnitine-independent. Using control and carnitine palmitoyltransferase 2 (CPT2)- and carnitine/acylcarnitine translocase (CACT)-deficient human fibroblasts, we investigated the oxidation of lauric acid (C12:0). Measurement of the acylcarnitine profile in the extracellular medium revealed significantly elevated levels of extracellular C10- and C12-carnitine in CPT2- and CACT-deficient fibroblasts. The accumulation of C12-carnitine indicates that lauric acid also uses the carnitine shuttle to access mitochondria. Moreover, the accumulation of extracellular C10-carnitine in CPT2- and CACT-deficient cells suggests an extramitochondrial pathway for the oxidation of lauric acid. Indeed, in the absence of peroxisomes C10-carnitine is not produced, proving that this intermediate is a product of peroxisomal ß-oxidation. In conclusion, when the carnitine shuttle is impaired lauric acid is partly oxidized in peroxisomes. This peroxisomal oxidation could be a compensatory mechanism to metabolize straight medium- and long-chain fatty acids, especially in cases of mitochondrial fatty acid ß-oxidation deficiency or overload.


Assuntos
Carnitina Aciltransferases/fisiologia , Carnitina O-Palmitoiltransferase/fisiologia , Carnitina/análogos & derivados , Carnitina/metabolismo , Fibroblastos/metabolismo , Erros Inatos do Metabolismo Lipídico/metabolismo , Peroxissomos/metabolismo , Pele/metabolismo , Carnitina Aciltransferases/deficiência , Carnitina Aciltransferases/metabolismo , Células Cultivadas , Fibroblastos/citologia , Imunofluorescência , Humanos , Ácidos Láuricos/química , Erros Inatos do Metabolismo Lipídico/patologia , Oxirredução , Pele/citologia
14.
FASEB J ; 27(5): 2039-44, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23322164

RESUMO

Acylcarnitines are commonly used in the diagnosis of mitochondrial fatty acid ß-oxidation disorders (mFAODs). It is generally assumed that this plasma acylcarnitine profile reflects the mitochondrial accumulation of acyl-CoAs. The identity of the enzymes and the mitochondrial and plasmalemmal transporters involved in the synthesis and export of these metabolites have remained undefined. We used lentiviral shRNA to knock down the expression of medium-chain acyl-CoA dehydrogenase (MCAD) in control and carnitine palmitoyltransferase 2 (CPT2)-, carnitine/acylcarnitine translocase (CACT)-, and plasmalemmal carnitine transporter (OCTN2)-deficient human fibroblasts. These cell lines, including mock-transduced controls, were loaded with decanoic acid and carnitine, followed by the measurement of the acylcarnitine profile in the extracellular medium. In control fibroblasts, MCAD knockdown markedly increased the production of octanoylcarnitine (3-fold, P<0.01). OCTN2-deficient cell lines also showed extracellular accumulation of octanoylcarnitine (2.8-fold, P<0.01), suggesting that the cellular export of acylcarnitines does not depend on OCTN2. In contrast, in CPT2- and CACT-deficient cells, the accumulation of octanoylcarnitine in the medium did not significantly increase in the MCAD knockdown. Similar results were obtained using pharmacological inhibition of CPT2 in fibroblasts from MCAD-deficient individuals. This shows that CPT2 and CACT are crucial for mitochondrial acylcarnitine formation and export to the extracellular fluids in mFAOD.


Assuntos
Carnitina Aciltransferases/metabolismo , Carnitina O-Palmitoiltransferase/metabolismo , Carnitina/análogos & derivados , Doenças Mitocondriais/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Acil-CoA Desidrogenase/genética , Acil-CoA Desidrogenase/metabolismo , Carnitina/metabolismo , Carnitina Aciltransferases/deficiência , Carnitina O-Palmitoiltransferase/deficiência , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Membro 5 da Família 22 de Carreadores de Soluto
15.
J Inherit Metab Dis ; 37(5): 709-14, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24531926

RESUMO

Inherited disorders of acyl-CoA metabolism, such as defects in amino acid metabolism and fatty acid oxidation can present with severe clinical symptoms either neonatally or later in life, but the pathophysiological mechanisms are often incompletely understood. We now report the discovery of a novel biochemical mechanism that could contribute to the pathophysiology of these disorders. We identified increased protein lysine butyrylation in short-chain acyl-CoA dehydrogenase (SCAD) deficient mice as a result of the accumulation of butyryl-CoA. Similarly, in SCAD deficient fibroblasts, lysine butyrylation was increased. Furthermore, malonyl-CoA decarboxylase (MCD) deficient patient cells had increased levels of malonylated lysines and propionyl-CoA carboxylase (PCC) deficient patient cells had increased propionylation of lysines. Since lysine acylation can greatly impact protein function, aberrant lysine acylation in inherited disorders associated with acyl-CoA accumulation may well play a role in their disease pathophysiology.


Assuntos
Acil Coenzima A/metabolismo , Acilação/genética , Erros Inatos do Metabolismo Lipídico/metabolismo , Proteínas/metabolismo , Acil-CoA Desidrogenase/deficiência , Animais , Linhagem Celular , Ácidos Graxos/metabolismo , Humanos , Erros Inatos do Metabolismo Lipídico/genética , Lisina/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias Hepáticas/metabolismo
16.
FASEB J ; 26(10): 4316-26, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22782973

RESUMO

Mitochondrial enoyl-CoA isomerase (ECI1) is an auxiliary enzyme involved in unsaturated fatty acid oxidation. In contrast to most of the other enzymes involved in fatty acid oxidation, a deficiency of ECI1 has yet to be identified in humans. We used wild-type (WT) and Eci1-deficient knockout (KO) mice to explore a potential presentation of human ECI1 deficiency. Upon food withdrawal, Eci1-deficient mice displayed normal blood ß-hydroxybutyrate levels (WT 1.09 mM vs. KO 1.10 mM), a trend to lower blood glucose levels (WT 4.58 mM vs. KO 3.87 mM, P=0.09) and elevated blood levels of unsaturated acylcarnitines, in particular C12:1 acylcarnitine (WT 0.03 µM vs. KO 0.09 µM, P<0.01). Feeding an olive oil-rich diet induced an even greater increase in C12:1 acylcarnitine levels (WT 0.01 µM vs. KO 0.04 µM, P<0.01). Overall, the phenotypic presentation of Eci1-deficient mice is mild, possibly caused by the presence of a second enoyl-CoA isomerase (Eci2) in mitochondria. Knockdown of Eci2 in Eci1-deficient fibroblasts caused a more pronounced accumulation of C12:1 acylcarnitine on incubation with unsaturated fatty acids (12-fold, P<0.05). We conclude that Eci2 compensates for Eci1 deficiency explaining the mild phenotype of Eci1-deficient mice. Hypoglycemia and accumulation of C12:1 acylcarnitine might be diagnostic markers to identify ECI1 deficiency in humans.


Assuntos
Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Ácidos Graxos Insaturados/metabolismo , Mitocôndrias/enzimologia , Animais , Glicemia/metabolismo , Isomerases de Ligação Dupla Carbono-Carbono/genética , Carnitina/análogos & derivados , Carnitina/sangue , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Dodecenoil-CoA Isomerase , Immunoblotting , Espectrometria de Massas , Camundongos , Camundongos Knockout , Oxirredução , Reação em Cadeia da Polimerase em Tempo Real
17.
Biochim Biophys Acta ; 1811(7-8): 441-51, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21621638

RESUMO

Imbalance in the supply and utilization of fatty acids (FA) is thought to contribute to intrahepatic lipid (IHL) accumulation in obesity. The aim of this study was to determine the time course of changes in the liver capacity to oxidize and store FA in response to high-fat diet (HFD). Adult male Wistar rats were fed either normal chow or HFD for 2.5weeks (short-term) and 25weeks (long-term). Short-term HFD feeding led to a 10% higher palmitoyl-l-carnitine-driven ADP-stimulated (state 3) oxygen consumption rate in isolated liver mitochondria indicating up-regulation of ß-oxidation. This adaptation was insufficient to cope with the dietary FA overload, as indicated by accumulation of long-chain acylcarnitines, depletion of free carnitine and increase in FA content in the liver, reflecting IHL accumulation. The latter was confirmed by in vivo((1))H magnetic resonance spectroscopy and Oil Red O staining. Long-term HFD feeding caused further up-regulation of mitochondrial ß-oxidation (24% higher oxygen consumption rate in state 3 with palmitoyl-l-carnitine as substrate) and stimulation of mitochondrial biogenesis as indicated by 62% higher mitochondrial DNA copy number compared to controls. These adaptations were paralleled by a partial restoration of free carnitine levels and a decrease in long-chain acylcarnitine content. Nevertheless, there was a further increase in IHL content, accompanied by accumulation of lipid peroxidation and protein oxidation products. In conclusion, partially effective adaption of hepatic FA metabolism to long-term HFD feeding came at a price of increased oxidative stress, caused by a combination of higher FA oxidation capacity and oversupply of FA.


Assuntos
Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/efeitos adversos , Ácidos Graxos/metabolismo , Fígado/metabolismo , Animais , Carnitina/análogos & derivados , Carnitina/metabolismo , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Metabolismo dos Lipídeos , Peroxidação de Lipídeos , Masculino , Mitocôndrias Hepáticas/metabolismo , Hepatopatia Gordurosa não Alcoólica , Obesidade/complicações , Obesidade/metabolismo , Estresse Oxidativo , Consumo de Oxigênio , Ratos , Ratos Wistar , Fatores de Tempo
18.
Mol Genet Metab ; 93(4): 403-10, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18077198

RESUMO

The pathogenesis of hypoketotic hypoglycemia and cardiomyopathy in patients with fatty acid oxidation (FAO) disorders is still poorly understood. In vitro studies are hampered by the lack of natural mutants to asses the effect of FAO inhibition. In addition, only a few inhibitors of FAO are known. Furthermore, most inhibitors of FAO are activating ligands of peroxisome proliferator-activated receptors (PPARs). We show that l-aminocarnitine (L-AC), a carnitine analog, inhibits FAO efficiently, but does not activate PPAR. L-AC inhibits carnitine palmitoyltransferase (CPT) with different sensitivities towards CPT1 and CPT2, as well as carnitine acylcarnitine translocase (CACT). We further characterized L-AC using fibroblasts cell lines from controls and patients with different FAO defects. In these cell lines acylcarnitine profiles were determined in culture medium after loading with [U-(13)C]palmitic acid. In control fibroblasts, L-AC inhibits FAO leading to a reduction of C2-acylcarnitine and elevation of C16-acylcarnitine. In very long-chain acyl-CoA dehydrogenase (VLCAD)-deficient fibroblasts, L-AC decreased the elevated C14-acylcarnitine and increased C16-acylcarnitine. In CACT and CPT2-deficient cell lines, L-AC did not change the already elevated C16-acylcarnitine level, showing that CPT1 is not inhibited. Oxidation of pristanic acid was only partly inhibited at high L-AC concentrations, indicating minimal CACT inhibition. Therefore, we conclude that in intact cells L-AC inhibits CPT2. Combined with our observation that l-AC does not activate PPAR, we suggest that L-AC is useful to simulate a FAO defect in cells from different origin.


Assuntos
Betaína/análogos & derivados , Carnitina/farmacologia , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Acetil-CoA C-Aciltransferase/metabolismo , Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Betaína/metabolismo , Betaína/farmacologia , Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Carnitina/metabolismo , Carnitina Aciltransferases/deficiência , Carnitina O-Palmitoiltransferase/antagonistas & inibidores , Carnitina O-Palmitoiltransferase/deficiência , Células Cultivadas , Enoil-CoA Hidratase/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Receptores Ativados por Proliferador de Peroxissomo/efeitos dos fármacos , Racemases e Epimerases/metabolismo , Síndrome de Zellweger/metabolismo
19.
J Med Genet ; 44(2): 153-60, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16963483

RESUMO

BACKGROUND: Usher syndrome, a combination of retinitis pigmentosa (RP) and sensorineural hearing loss with or without vestibular dysfunction, displays a high degree of clinical and genetic heterogeneity. Three clinical subtypes can be distinguished, based on the age of onset and severity of the hearing impairment, and the presence or absence of vestibular abnormalities. Thus far, eight genes have been implicated in the syndrome, together comprising 347 protein-coding exons. METHODS: To improve DNA diagnostics for patients with Usher syndrome, we developed a genotyping microarray based on the arrayed primer extension (APEX) method. Allele-specific oligonucleotides corresponding to all 298 Usher syndrome-associated sequence variants known to date, 76 of which are novel, were arrayed. RESULTS: Approximately half of these variants were validated using original patient DNAs, which yielded an accuracy of >98%. The efficiency of the Usher genotyping microarray was tested using DNAs from 370 unrelated European and American patients with Usher syndrome. Sequence variants were identified in 64/140 (46%) patients with Usher syndrome type I, 45/189 (24%) patients with Usher syndrome type II, 6/21 (29%) patients with Usher syndrome type III and 6/20 (30%) patients with atypical Usher syndrome. The chip also identified two novel sequence variants, c.400C>T (p.R134X) in PCDH15 and c.1606T>C (p.C536S) in USH2A. CONCLUSION: The Usher genotyping microarray is a versatile and affordable screening tool for Usher syndrome. Its efficiency will improve with the addition of novel sequence variants with minimal extra costs, making it a very useful first-pass screening tool.


Assuntos
Análise de Sequência com Séries de Oligonucleotídeos , Síndromes de Usher/genética , DNA/genética , Primers do DNA , Europa (Continente) , Variação Genética , Genótipo , Humanos
20.
Otol Neurotol ; 26(1): 52-8, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15699719

RESUMO

OBJECTIVE: Genotype a family trait with autosomal dominant nonsyndromic sensorineural hearing impairment guided only by the phenotype. STUDY DESIGN: Family study. SETTING: Tertiary referral center. PATIENTS: Fifteen family members. METHODS: In the first phase, sequence analysis was performed on DNA isolated from buccal swabs of the proband and her daughter, guided by the phenotype based on audiometric data that were already available. After detection of the W276S missense mutation in the KCNQ4 gene in both patients, this finding was confirmed in the other affected family members. All participants completed a questionnaire, were clinically examined, and underwent standard pure-tone audiometry. The results were analyzed to refine the phenotypic features of the family trait. RESULTS: All clinically affected participants were carriers of the W276S hotspot mutation in exon 5 of the KCNQ4 gene on chromosome 1p34. Refined phenotypic features confirmed previously described phenotypes of DFNA2 families. CONCLUSIONS: Phenotype determination can be cost saving and very effective in detecting the genotype of autosomal dominant nonsyndromic hearing impairment, especially when phenotype analyses can be performed on data that are already available or easily collected.


Assuntos
Genótipo , Perda Auditiva de Alta Frequência/genética , Perda Auditiva Neurossensorial/genética , Mutação de Sentido Incorreto , Fenótipo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Audiometria de Tons Puros , Criança , Aberrações Cromossômicas , Comparação Transcultural , Análise Mutacional de DNA , Progressão da Doença , Feminino , Seguimentos , Genes Dominantes , Triagem de Portadores Genéticos , Perda Auditiva de Alta Frequência/diagnóstico , Perda Auditiva Neurossensorial/diagnóstico , Humanos , Canais de Potássio KCNQ , Masculino , Pessoa de Meia-Idade , Países Baixos , Linhagem , Presbiacusia/diagnóstico , Presbiacusia/genética , Análise de Sequência de DNA , Estatística como Assunto
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