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2.
Hum Mol Genet ; 28(20): 3339-3354, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31334547

RESUMO

Human dihydrolipoamide dehydrogenase (hLADH, hE3) deficiency (OMIM# 246900) is an often prematurely lethal genetic disease usually caused by inactive or partially inactive hE3 variants. Here we report the crystal structure of wild-type hE3 at an unprecedented high resolution of 1.75 Å and the structures of six disease-causing hE3 variants at resolutions ranging from 1.44 to 2.34 Å. P453L proved to be the most deleterious substitution in structure as aberrations extensively compromised the active site. The most prevalent G194C-hE3 variant primarily exhibited structural alterations close to the substitution site, whereas the nearby cofactor-binding residues were left unperturbed. The G426E substitution mainly interfered with the local charge distribution introducing dynamics to the substitution site in the dimer interface; G194C and G426E both led to minor structural changes. The R460G, R447G and I445M substitutions all perturbed a solvent accessible channel, the so-called H+/H2O channel, leading to the active site. Molecular pathomechanisms of enhanced reactive oxygen species (ROS) generation and impaired binding to multienzyme complexes were also addressed according to the structural data for the relevant mutations. In summary, we present here for the first time a comprehensive study that links three-dimensional structures of disease-causing hE3 variants to residual hLADH activities, altered capacities for ROS generation, compromised affinities for multienzyme complexes and eventually clinical symptoms. Our results may serve as useful starting points for future therapeutic intervention approaches.


Assuntos
Di-Hidrolipoamida Desidrogenase/deficiência , Complexos Multienzimáticos/metabolismo , Domínio Catalítico , Di-Hidrolipoamida Desidrogenase/genética , Humanos , Complexos Multienzimáticos/genética , Mutagênese Sítio-Dirigida , Mutação/genética , Conformação Proteica , Espécies Reativas de Oxigênio/metabolismo
3.
FASEB J ; 27(6): 2392-406, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23475850

RESUMO

A decline in α-ketoglutarate dehydrogenase complex (KGDHC) activity has been associated with neurodegeneration. Provision of succinyl-CoA by KGDHC is essential for generation of matrix ATP (or GTP) by substrate-level phosphorylation catalyzed by succinyl-CoA ligase. Here, we demonstrate ATP consumption in respiration-impaired isolated and in situ neuronal somal mitochondria from transgenic mice with a deficiency of either dihydrolipoyl succinyltransferase (DLST) or dihydrolipoyl dehydrogenase (DLD) that exhibit a 20-48% decrease in KGDHC activity. Import of ATP into the mitochondrial matrix of transgenic mice was attributed to a shift in the reversal potential of the adenine nucleotide translocase toward more negative values due to diminished matrix substrate-level phosphorylation, which causes the translocase to reverse prematurely. Immunoreactivity of all three subunits of succinyl-CoA ligase and maximal enzymatic activity were unaffected in transgenic mice as compared to wild-type littermates. Therefore, decreased matrix substrate-level phosphorylation was due to diminished provision of succinyl-CoA. These results were corroborated further by the finding that mitochondria from wild-type mice respiring on substrates supporting substrate-level phosphorylation exhibited ~30% higher ADP-ATP exchange rates compared to those obtained from DLST(+/-) or DLD(+/-) littermates. We propose that KGDHC-associated pathologies are a consequence of the inability of respiration-impaired mitochondria to rely on "in-house" mitochondrial ATP reserves.


Assuntos
Aciltransferases/deficiência , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Di-Hidrolipoamida Desidrogenase/deficiência , Complexo Cetoglutarato Desidrogenase/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/genética , Animais , Di-Hidrolipoamida Desidrogenase/genética , Di-Hidrolipoamida Desidrogenase/metabolismo , Feminino , Complexo Cetoglutarato Desidrogenase/química , Complexo Cetoglutarato Desidrogenase/deficiência , Complexo Cetoglutarato Desidrogenase/genética , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação , Especificidade por Substrato
4.
Cell Host Microbe ; 9(1): 21-31, 2011 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-21238944

RESUMO

Mycobacterium tuberculosis (Mtb) adapts to persist in a nutritionally limited macrophage compartment. Lipoamide dehydrogenase (Lpd), the third enzyme (E3) in Mtb's pyruvate dehydrogenase complex (PDH), also serves as E1 of peroxynitrite reductase/peroxidase (PNR/P), which helps Mtb resist host-reactive nitrogen intermediates. In contrast to Mtb lacking dihydrolipoamide acyltransferase (DlaT), the E2 of PDH and PNR/P, Lpd-deficient Mtb is severely attenuated in wild-type and immunodeficient mice. This suggests that Lpd has a function that DlaT does not share. When DlaT is absent, Mtb upregulates an Lpd-dependent branched-chain keto acid dehydrogenase (BCKADH) encoded by pdhA, pdhB, pdhC, and lpdC. Without Lpd, Mtb cannot metabolize branched-chain amino acids and potentially toxic branched-chain intermediates accumulate. Mtb deficient in both DlaT and PdhC phenocopies Lpd-deficient Mtb. Thus, Mtb critically requires BCKADH along with PDH and PNR/P for pathogenesis. These findings position Lpd as a potential target for anti-infectives against Mtb.


Assuntos
Di-Hidrolipoamida Desidrogenase/metabolismo , Mycobacterium tuberculosis/patogenicidade , Fatores de Virulência/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Animais , Carga Bacteriana , Di-Hidrolipoamida Desidrogenase/deficiência , Modelos Animais de Doenças , Pulmão/microbiologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multienzimáticos/deficiência , Complexos Multienzimáticos/metabolismo , Baço/microbiologia , Tuberculose/microbiologia , Tuberculose/patologia , Virulência , Fatores de Virulência/deficiência
5.
J Mol Med (Berl) ; 89(2): 161-70, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21079907

RESUMO

Medicine today offers no cure for patients suffering from mitochondrial disorders, such as lipoamide dehydrogenase (LAD; also known as E3) deficiency, and treatment is limited to symptomatic care. LAD is one of the components of the α-ketoacid dehydrogenase complexes, which are mitochondrial multienzyme complexes crucial for the metabolism of carbohydrates and amino acids. Recently, we tested the therapeutic approach for treating mitochondrial disorders whereby the activity of multicomponent complexes in the mitochondria is restored by TAT-mediated enzyme replacement therapy (ERT). The LAD deficiency disease was used before as a proof-of-principle in vitro, in patients' cells, utilizing the TAT-LAD fusion protein. In this report, we present successful TAT-mediated ERT in an in vivo mouse model using E3-deficient mice. We demonstrate the delivery of TAT-LAD into E3-deficient mice tissues and that a single administration of TAT-LAD results in a significant increase in the enzymatic activity of the mitochondrial multienzyme complex pyruvate dehydrogenase complex within the liver, heart and, most importantly, the brain of TAT-LAD-treated E3-deficient mice. We believe that this TAT-mediated ERT approach could change the management of mitochondrial disorders and of other metabolic diseases in modern medicine.


Assuntos
Di-Hidrolipoamida Desidrogenase/deficiência , Terapia de Reposição de Enzimas , Produtos do Gene tat/metabolismo , Mitocôndrias/enzimologia , Proteínas Recombinantes de Fusão , Animais , Di-Hidrolipoamida Desidrogenase/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Especificidade de Órgãos/genética , Complexo Piruvato Desidrogenase/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia
6.
Neuroscience ; 153(4): 986-96, 2008 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-18423880

RESUMO

Mitochondrial defects including reduction of a key mitochondrial tricarboxylic acid cycle enzyme alpha-ketoglutarate-dehydrogenase complex (KGDHC) are characteristic of many neurodegenerative diseases. KGDHC consists of alpha-ketoglutarate dehydrogenase, dihydrolipoyl succinyltransferase (E2k), and dihydrolipoamide dehydrogenase (Dld) subunits. We investigated whether Dld or E2k deficiency influences adult brain neurogenesis using immunohistochemistry for the immature neuron markers, doublecortin (Dcx) and polysialic acid-neural cell adhesion molecule, as well as a marker for proliferation, proliferating cell nuclear antigen (PCNA). Both Dld- and E2k-deficient mice showed reduced Dcx-positive neuroblasts in the subgranular zone (SGZ) of the hippocampal dentate gyrus compared with wild-type mice. In the E2k knockout mice, increased immunoreactivity for the lipid peroxidation marker, malondialdehyde occurred in the SGZ. These alterations did not occur in the subventricular zone (SVZ). PCNA staining revealed decreased proliferation in the SGZ of E2k-deficient mice. In a transgenic mouse model of Alzheimer's disease, Dcx-positive cells in the SGZ were also reduced compared with wild type, but Dld deficiency did not exacerbate the reduction. In the malonate lesion model of Huntington's disease, Dld deficiency did not alter the lesion-induced increase and migration of Dcx-positive cells from the SVZ into the ipsilateral striatum. Thus, the KGDHC subunit deficiencies associated with elevated lipid peroxidation selectively reduced the number of neuroblasts and proliferating cells in the hippocampal neurogenic zone. However, these mitochondrial defects neither exacerbated certain pathological conditions, such as amyloid precursor protein (APP) mutation-induced reduction of SGZ neuroblasts, nor inhibited malonate-induced migration of SVZ neuroblasts. Our findings support the view that mitochondrial dysfunction can influence the number of neural progenitor cells in the hippocampus of adult mice.


Assuntos
Encéfalo/patologia , Proliferação de Células , Mitocôndrias/enzimologia , Doenças Neurodegenerativas/enzimologia , Doenças Neurodegenerativas/patologia , Neurônios/ultraestrutura , Aciltransferases/deficiência , Análise de Variância , Animais , Di-Hidrolipoamida Desidrogenase/deficiência , Modelos Animais de Doenças , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Regulação Enzimológica da Expressão Gênica/genética , Peroxidação de Lipídeos/genética , Malondialdeído/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Doenças Neurodegenerativas/genética , Neurônios/metabolismo , Neurônios/patologia , Neuropeptídeos/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Coloração pela Prata/métodos
8.
Mol Ther ; 16(4): 691-7, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18362926

RESUMO

Modern medicine offers no cure for mitochondrial disorders such as lipoamide dehydrogenase (LAD) deficiency. LAD is the E3 subunit shared by alpha-ketoacid dehydrogenase complexes in the mitochondrial matrix, and these complexes are crucial for the metabolism of carbohydrates and amino acids. We propose a novel concept for restoring the activity of an immense multicomponent enzymatic complex by replacing one mutated component, the LAD subunit. Our approach entails the fusing of LAD with the transactivator of transcription (TAT) peptide, which is capable of rapidly crossing biological membranes, thereby allowing TAT-LAD to be delivered into cells and their mitochondria where it can replace the mutated endogenous enzyme. Our results show that TAT-LAD is indeed delivered into the cells and their mitochondria, where it is processed, restoring LAD activity to normal values and, most importantly, increasing the activity of pyruvate dehydrogenase complex. We report here, for the first time, that TAT-mediated replacement of one mutated component restores the activity of an essential mitochondrial multicomponent enzymatic complex in cells of patients with enzyme deficiencies. We believe that this approach involving TAT-mediated enzyme replacement therapy (ERT) can be applied to the treatment of LAD deficiency as well as to other mitochondrial and metabolic disorders.


Assuntos
Di-Hidrolipoamida Desidrogenase/deficiência , Di-Hidrolipoamida Desidrogenase/genética , Mitocôndrias/efeitos dos fármacos , Fragmentos de Peptídeos/genética , Proteínas Recombinantes de Fusão/uso terapêutico , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Células Cultivadas , Di-Hidrolipoamida Desidrogenase/metabolismo , Ativação Enzimática , Humanos , Mitocôndrias/enzimologia , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo
9.
J Biomed Sci ; 15(1): 37-46, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17960497

RESUMO

Human dihydrolipoamide dehydrogenase (hE3) is a common component of alpha-ketoacid dehydrogenase complexes. Mutations of this homodimeric protein cause E3 deficiency and are always fatal. To investigate its reaction mechanism, we first performed multiple sequence alignment with other 17 eukaryotic E3s. According to hE3 structure and the result of multiple sequence alignment, two amino acids, T148 and R281, were subjected to mutagenesis and four hE3 mutants, T148G, T148S, R281N, and R281K, were expressed and assayed. The specific activities of T148G, T148S, R281N, and R281K are 76.34%, 88.62%, 12.50%, and 11.93% to that of wild-type E3, respectively. The FAD content analysis indicated that the FAD content of these mutant E3s were about 71.0%, 92%, 96%, and 93% that of wild-type E3, respectively. The molecular weight analysis showed that these three mutant proteins form the dimer. Kinetic data demonstrated that the K(cat) of forward reaction of all mutants, except T148 mutants, were decreased dramatically. The results of kinetic study suggest that T148 is not important to E3 catalytic function and R281 play a role in the catalytic function of the E3.


Assuntos
Di-Hidrolipoamida Desidrogenase/química , Di-Hidrolipoamida Desidrogenase/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Domínio Catalítico/genética , Primers do DNA/genética , Di-Hidrolipoamida Desidrogenase/deficiência , Di-Hidrolipoamida Desidrogenase/metabolismo , Dimerização , Flavina-Adenina Dinucleotídeo/análise , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Mutagênese Sítio-Dirigida , NAD/metabolismo , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos
10.
Am J Med Genet A ; 140(14): 1542-52, 2006 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-16770810

RESUMO

We have diagnosed dihydrolipoamide dehydrogenase (DLD) deficiency in two male second cousins, who presented with markedly different clinical phenotypes. Patient 1 had a recurrent encephalopathy, and patient 2 had microcephaly and lactic acidosis. Their presentation is unusual, in that the DLD subunit deficiency had little effect on pyruvate dehydrogenase complex activity, but caused a severe reduction in the activities of other enzymes that utilize this subunit. We have identified two mutations in the DLD gene in each patient. The second cousins have one novel mutation in common resulting in a substitution of isoleucine for threonine (I47T), which has not been previously reported in the literature. Patient 1 has a second mutation that has been reported to be common in the Ashkenazi Jewish population, G229C. Patient 2 has a second mutation, E375K, which has also been previously reported in the literature. Enzyme kinetic measurements on patient fibroblasts show that under certain conditions, one heteroallelic mutation may have a higher K(m). This may account for the differing clinical phenotypes. These findings have important repercussions for other patients with similar clinical phenotypes, as DLD activity is not normally measured in cases with normal PDHc activity.


Assuntos
Di-Hidrolipoamida Desidrogenase/deficiência , Di-Hidrolipoamida Desidrogenase/genética , Mutação Puntual , Complexo Piruvato Desidrogenase/metabolismo , Acidose Láctica/enzimologia , Acidose Láctica/genética , Substituição de Aminoácidos , Sequência de Bases , Encefalopatias Metabólicas/enzimologia , Encefalopatias Metabólicas/genética , Criança , Pré-Escolar , DNA/genética , Di-Hidrolipoamida Desidrogenase/química , Feminino , Fibroblastos/enzimologia , Heterozigoto , Humanos , Técnicas In Vitro , Cinética , Masculino , Microcefalia/enzimologia , Microcefalia/genética , Modelos Moleculares , Fenótipo , Subunidades Proteicas , Complexo Piruvato Desidrogenase/química , Complexo Piruvato Desidrogenase/genética
11.
J Inherit Metab Dis ; 29(1): 203-4, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16601893

RESUMO

A late-onset presentation of lipoamide dehydrogenase (E3) deficiency is described in a North American Ashkenazi Jewish (AJ) family. Diagnosis was made by urine organic acid and molecular analyses.


Assuntos
Di-Hidrolipoamida Desidrogenase/deficiência , Erros Inatos do Metabolismo/diagnóstico , Idade de Início , Pré-Escolar , Di-Hidrolipoamida Desidrogenase/genética , Éxons , Heterozigoto , Humanos , Judeus , Masculino , Erros Inatos do Metabolismo/etnologia , Erros Inatos do Metabolismo/urina , Mutação , América do Norte
12.
J Mol Biol ; 350(3): 543-52, 2005 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-15946682

RESUMO

Human dihydrolipoamide dehydrogenase (hE3) is an enzymatic component common to the mitochondrial alpha-ketoacid dehydrogenase and glycine decarboxylase complexes. Mutations to this homodimeric flavoprotein cause the often-fatal human disease known as E3 deficiency. To catalyze the oxidation of dihydrolipoamide, hE3 uses two molecules: non-covalently bound FAD and a transiently bound substrate, NAD+. To address the catalytic mechanism of hE3 and the structural basis for E3 deficiency, the crystal structures of hE3 in the presence of NAD+ or NADH have been determined at resolutions of 2.5A and 2.1A, respectively. Although the overall fold of the enzyme is similar to that of yeast E3, these two structures differ at two loops that protrude from the proteins and at their FAD-binding sites. The structure of oxidized hE3 with NAD+ bound demonstrates that the nicotinamide moiety is not proximal to the FAD. When NADH is present, however, the nicotinamide base stacks directly on the isoalloxazine ring system of the FAD. This is the first time that this mechanistically requisite conformation of NAD+ or NADH has been observed in E3 from any species. Because E3 structures were previously available only from unicellular organisms, speculations regarding the molecular mechanisms of E3 deficiency were based on homology models. The current hE3 structures show directly that the disease-causing mutations occur at three locations in the human enzyme: the dimer interface, the active site, and the FAD and NAD(+)-binding sites. The mechanisms by which these mutations impede the function of hE3 are discussed.


Assuntos
Di-Hidrolipoamida Desidrogenase/química , Di-Hidrolipoamida Desidrogenase/deficiência , Mutação , Sítios de Ligação , Catálise , Clonagem Molecular , Cristalografia por Raios X , Bases de Dados de Proteínas , Dimerização , Dissulfetos , Glicina/química , Humanos , Doença da Urina de Xarope de Bordo/genética , Doença da Urina de Xarope de Bordo/metabolismo , Modelos Moleculares , NAD/química , Niacinamida/química , Oxigênio/metabolismo , Potyvirus/metabolismo , Ligação Proteica , Pseudomonas putida/metabolismo , Saccharomyces cerevisiae/metabolismo , Ácido Tióctico/metabolismo
13.
J Neurosci ; 24(36): 7779-88, 2004 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-15356189

RESUMO

Mitochondria-produced reactive oxygen species (ROS) are thought to contribute to cell death caused by a multitude of pathological conditions. The molecular sites of mitochondrial ROS production are not well established but are generally thought to be located in complex I and complex III of the electron transport chain. We measured H(2)O(2) production, respiration, and NADPH reduction level in rat brain mitochondria oxidizing a variety of respiratory substrates. Under conditions of maximum respiration induced with either ADP or carbonyl cyanide p-trifluoromethoxyphenylhydrazone,alpha-ketoglutarate supported the highest rate of H(2)O(2) production. In the absence of ADP or in the presence of rotenone, H(2)O(2) production rates correlated with the reduction level of mitochondrial NADPH with various substrates, with the exception of alpha-ketoglutarate. Isolated mitochondrial alpha-ketoglutarate dehydrogenase (KGDHC) and pyruvate dehydrogenase (PDHC) complexes produced superoxide and H(2)O(2). NAD(+) inhibited ROS production by the isolated enzymes and by permeabilized mitochondria. We also measured H(2)O(2) production by brain mitochondria isolated from heterozygous knock-out mice deficient in dihydrolipoyl dehydrogenase (Dld). Although this enzyme is a part of both KGDHC and PDHC, there was greater impairment of KGDHC activity in Dld-deficient mitochondria. These mitochondria also produced significantly less H(2)O(2) than mitochondria isolated from their littermate wild-type mice. The data strongly indicate that KGDHC is a primary site of ROS production in normally functioning mitochondria.


Assuntos
Antimicina A/análogos & derivados , Complexo Cetoglutarato Desidrogenase/metabolismo , Mitocôndrias/enzimologia , Proteínas do Tecido Nervoso/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ubiquinona/análogos & derivados , Difosfato de Adenosina/farmacologia , Animais , Antimicina A/farmacologia , Coenzimas , Di-Hidrolipoamida Desidrogenase/deficiência , Di-Hidrolipoamida Desidrogenase/genética , Di-Hidrolipoamida Desidrogenase/metabolismo , Transporte de Elétrons/efeitos dos fármacos , Transporte de Elétrons/fisiologia , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Peróxido de Hidrogênio/metabolismo , Membranas Intracelulares/fisiologia , Ácidos Cetoglutáricos/metabolismo , Potenciais da Membrana , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , NAD/metabolismo , NADP/metabolismo , Oligomicinas/farmacologia , Oxirredução , Prosencéfalo/enzimologia , Prosencéfalo/ultraestrutura , Complexo Piruvato Desidrogenase/metabolismo , Ratos , Ratos Sprague-Dawley , Rotenona/farmacologia , Ácido Succínico/metabolismo , Superóxido Dismutase/farmacologia , Superóxidos/metabolismo , Ubiquinona/análise
14.
J Neurochem ; 88(6): 1352-60, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15009635

RESUMO

Altered energy metabolism, including reductions in activities of the key mitochondrial enzymes alpha-ketoglutarate dehydrogenase complex (KGDHC) and pyruvate dehydrogenase complex (PDHC), are characteristic of many neurodegenerative disorders including Alzheimer's Disease (AD), Parkinson's disease (PD) and Huntington's disease (HD). Dihydrolipoamide dehydrogenase is a critical subunit of KGDHC and PDHC. We tested whether mice that are deficient in dihydrolipoamide dehydrogenase (Dld+/-) show increased vulnerability to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), malonate and 3-nitropropionic acid (3-NP), which have been proposed for use in models of PD and HD. Administration of MPTP resulted in significantly greater depletion of tyrosine hydroxylase-positive neurons in the substantia nigra of Dld+/- mice than that seen in wild-type littermate controls. Striatal lesion volumes produced by malonate and 3-NP were significantly increased in Dld+/- mice. Studies of isolated brain mitochondria treated with 3-NP showed that both succinate-supported respiration and membrane potential were suppressed to a greater extent in Dld+/- mice. KGDHC activity was also found to be reduced in putamen from patients with HD. These findings provide further evidence that mitochondrial defects may contribute to the pathogenesis of neurodegenerative diseases.


Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , Di-Hidrolipoamida Desidrogenase/deficiência , Predisposição Genética para Doença , Malonatos , Doenças Neurodegenerativas/enzimologia , Propionatos , Animais , Núcleo Caudado/efeitos dos fármacos , Núcleo Caudado/enzimologia , Núcleo Caudado/patologia , Contagem de Células , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , Córtex Cerebral/patologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/enzimologia , Corpo Estriado/patologia , Di-Hidrolipoamida Desidrogenase/genética , Modelos Animais de Doenças , Heterozigoto , Doença de Huntington/induzido quimicamente , Doença de Huntington/enzimologia , Doença de Huntington/patologia , Complexo Cetoglutarato Desidrogenase/efeitos dos fármacos , Complexo Cetoglutarato Desidrogenase/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Doenças Neurodegenerativas/induzido quimicamente , Doenças Neurodegenerativas/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Nitrocompostos , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/enzimologia , Transtornos Parkinsonianos/patologia , Putamen/efeitos dos fármacos , Putamen/enzimologia , Putamen/patologia , Complexo Piruvato Desidrogenase/efeitos dos fármacos , Complexo Piruvato Desidrogenase/metabolismo , Substância Negra/efeitos dos fármacos , Substância Negra/enzimologia , Substância Negra/patologia
15.
Eur J Pediatr ; 162(10): 714-8, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12925875

RESUMO

UNLABELLED: A boy with recurrent episodes of hypoglycaemia and ataxia, microcephaly, mental retardation, permanent lactic acidaemia, intermittent 2-oxoglutaric aciduria as well as elevation of serum branched chain amino acids was diagnosed with dihydrolipoamide dehydrogenase (E3) deficiency. Analysis of genomic DNA revealed compound heterozygosity for two novel mutations: I393T in exon 11, located at the interface domain of the protein and possibly interfering with its dimerisation, and IVS9+1G>A located at a consensus splice site. A heterozygous polymorphism was also detected. In the patient's cDNA the I393T mutation and the polymorphism appeared to be homozygous, indicating that the mRNA coming from the IVS9+1G>A mutant allele is not stable. CONCLUSION: as opposed to the non-neurological phenotype of patients with a homozygous G229C mutation, this patient developed Leigh syndrome. Dihydrolipoamide dehydrogenase and pyruvate dehydrogenase complex activities in muscle were 29% and 14% of the lowest control values, respectively. Pyruvate dehydrogenase complex activity in fibroblasts was normal, however, indicating that the biochemical examination of defects in energy metabolism should be performed in a more energy demanding tissue.


Assuntos
Di-Hidrolipoamida Desidrogenase/deficiência , Di-Hidrolipoamida Desidrogenase/genética , Doença de Leigh/genética , Pré-Escolar , Fibroblastos/enzimologia , Heterozigoto , Humanos , Masculino , Músculo Esquelético/enzimologia , Mutação de Sentido Incorreto , Complexo Piruvato Desidrogenase/genética , Sítios de Splice de RNA
16.
Biochem Biophys Res Commun ; 269(2): 382-6, 2000 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-10708561

RESUMO

Lipoamide dehydrogenase deficiency is an inborn error of several metabolic pathways, including pyruvate metabolism, Krebs cycle, and branched-chain amino acid degradation. The clinical course is variable, ranging from infantile neurodegenerative disease to recurrent episodes of liver failure or myoglobinuria starting later in life. In contrast, residual enzymatic activity in muscle tissue spans over a narrow range. Despite the recent elucidation of the underlying molecular pathology in most patients, relationships between the genotype and the biochemical and clinical phenotype remain unclear. In order to find a suitable assay for the prediction of clinical outcome and assessment of treatment, we have evaluated enzymatic activities and energetic states in fibroblasts from lipoamide dehydrogenase-deficient patients representing three different phenotypes and genotypes. Direct relationships between clinical parameters such as age of onset and disease severity and biochemical characteristics, including lipoamide dehydrogenase activity, pyruvate dehydrogenase complex activity, and ATP production ratio in fibroblasts, were identified. Clinical parameters were not reflected by lactate/pyruvate ratio. ATP production rate was in direct relationship with the severity of the neurological involvement; the patient with reduced ATP synthesis to 30% of the control mean had a severe neurodegenerative disease, whereas ATP synthesis values above 45% were associated with a more favorable course. Incubation of the patients' fibroblasts with dichloroacetate coupled with thiamin resulted in slight but significant improvement of the cell energetic state.


Assuntos
Trifosfato de Adenosina/biossíntese , Di-Hidrolipoamida Desidrogenase/deficiência , Erros Inatos do Metabolismo/metabolismo , Adulto , Linhagem Celular , Di-Hidrolipoamida Desidrogenase/metabolismo , Humanos , Músculos/enzimologia , Estresse Oxidativo , Complexo Piruvato Desidrogenase/metabolismo
17.
Biochem Biophys Res Commun ; 262(1): 163-6, 1999 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-10448086

RESUMO

An infant with a neurodegenerative disorder accompanied by lactic acidemia is described. In muscle homogenate, the activity of lipoamide dehydrogenase (LAD), the third catalytic subunit of pyruvate dehydrogenase complex (PDHc), alpha-ketoglutarate dehydrogenase complex (KGDHc), and branched-chain keto acid dehydrogenase complex was reduced to 15% of the control. The activity of PDHc was undetectable and the activity of KGDHc was 2% of the control mean. The immunoreactive LAD protein was reduced to about 10% of the control. Direct sequencing of LAD cDNA revealed only one mutation, substituting Asp for Val at position 479 of the precursor form. The mutation resides within the interface domain and likely perturbs stable dimerization. The phenotypic heterogeneity in LAD deficiency is not directly correlated with the residual LAD activity but rather with its impact on the multienzymatic complex activity.


Assuntos
Di-Hidrolipoamida Desidrogenase/química , Di-Hidrolipoamida Desidrogenase/deficiência , Mutação , Sequência de Aminoácidos , Substituição de Aminoácidos , Di-Hidrolipoamida Desidrogenase/genética , Di-Hidrolipoamida Desidrogenase/metabolismo , Dimerização , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Feminino , Heterozigoto , Humanos , Lactente , Complexo Cetoglutarato Desidrogenase/química , Complexo Cetoglutarato Desidrogenase/deficiência , Complexo Cetoglutarato Desidrogenase/genética , Complexo Cetoglutarato Desidrogenase/metabolismo , Linfócitos/enzimologia , Masculino , Mitocôndrias Musculares/enzimologia , Dados de Sequência Molecular , Músculos/enzimologia , Músculos/patologia , Doenças Neurodegenerativas/enzimologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Fenótipo , Estrutura Secundária de Proteína , Complexo Piruvato Desidrogenase/química , Complexo Piruvato Desidrogenase/genética , Complexo Piruvato Desidrogenase/metabolismo , Doença da Deficiência do Complexo de Piruvato Desidrogenase/enzimologia , Doença da Deficiência do Complexo de Piruvato Desidrogenase/genética , Doença da Deficiência do Complexo de Piruvato Desidrogenase/patologia
18.
J Hepatol ; 29(3): 482-4, 1998 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9764998

RESUMO

Lipoamide dehydrogenase deficiency is a rare disease, manifested in early childhood by lactic acidemia, progressive neurological damage and death in most cases. We report a case of lipoamide dehydrogenase deficiency in a 34-year-old Ashkenazi-Jewish woman. The deficiency manifested as acute hepatitis without cognitive impairment or acidosis. The patient's brother also had lipoamide dehydrogenase deficiency, diagnosed at the age of 20, and manifested as hepatocellular damage, lactic acidemia and myoglobinuria. We assume that the trigger for this hepatocellular damage was prolonged fasting, and that otherwise the patient might have gone undiagnosed. Other cases in Ashkenazi Jews of mild lipoamide dehydrogenase deficiency with hepatocellular injury but without central nervous system involvement are reviewed.


Assuntos
Di-Hidrolipoamida Desidrogenase/deficiência , Hepatite/enzimologia , Doença Aguda , Adulto , Feminino , Humanos , Resultado do Tratamento
19.
Intensive Care Med ; 24(7): 706-8, 1998 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-9722041

RESUMO

We report on a young woman with congenital methaemoglobinaemia detected by a pulse oximeter during anaesthesia. Investigation of the patient and her family showed that the methaemoglobinaemia resulted from a recessive deficiency of NADH-diaphorase enzyme. A knowledge of the working principles and limitations of pulse oximetry is essential to determine appropriate management in desaturation episodes during the perioperative period.


Assuntos
Di-Hidrolipoamida Desidrogenase/deficiência , Metemoglobinemia/congênito , Metemoglobinemia/diagnóstico , Oximetria/normas , Adolescente , Anestesia , Feminino , Genes Recessivos , Humanos , Metemoglobinemia/genética , Metemoglobinemia/metabolismo , Monitorização Intraoperatória , Linhagem , Reprodutibilidade dos Testes
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