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1.
Proc Natl Acad Sci U S A ; 111(3): 960-5, 2014 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-24395786

RESUMO

Uncoupling protein 2 (UCP2) is involved in various physiological and pathological processes such as insulin secretion, stem cell differentiation, cancer, and aging. However, its biochemical and physiological function is still under debate. Here we show that UCP2 is a metabolite transporter that regulates substrate oxidation in mitochondria. To shed light on its biochemical role, we first studied the effects of its silencing on the mitochondrial oxidation of glucose and glutamine. Compared with wild-type, UCP2-silenced human hepatocellular carcinoma (HepG2) cells, grown in the presence of glucose, showed a higher inner mitochondrial membrane potential and ATP:ADP ratio associated with a lower lactate release. Opposite results were obtained in the presence of glutamine instead of glucose. UCP2 reconstituted in lipid vesicles catalyzed the exchange of malate, oxaloacetate, and aspartate for phosphate plus a proton from opposite sides of the membrane. The higher levels of citric acid cycle intermediates found in the mitochondria of siUCP2-HepG2 cells compared with those found in wild-type cells in addition to the transport data indicate that, by exporting C4 compounds out of mitochondria, UCP2 limits the oxidation of acetyl-CoA-producing substrates such as glucose and enhances glutaminolysis, preventing the mitochondrial accumulation of C4 metabolites derived from glutamine. Our work reveals a unique regulatory mechanism in cell bioenergetics and provokes a substantial reconsideration of the physiological and pathological functions ascribed to UCP2 based on its purported uncoupling properties.


Assuntos
Carbono/química , Glucose/metabolismo , Glutamina/metabolismo , Canais Iônicos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Oxigênio/química , Catálise , Respiração Celular/fisiologia , Ciclo do Ácido Cítrico , Metabolismo Energético , Inativação Gênica , Células HEK293 , Células Hep G2 , Humanos , Lipossomos/química , Potencial da Membrana Mitocondrial , Ácido Oxaloacético/metabolismo , Consumo de Oxigênio , Fosfatos/química , Proteína Desacopladora 2
2.
FEBS J ; 277(5): 1172-81, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20121944

RESUMO

The mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides and cofactors across the inner mitochondrial membrane. The genome of Drosophila melanogaster encodes at least 46 members of this family. Only five of these have been characterized, whereas the transport functions of the remainder cannot be assessed with certainty. In the present study, we report the functional identification of two D. melanogaster genes distantly related to the human and yeast thiamine pyrophosphate carrier (TPC) genes as well as the corresponding expression pattern throughout development. Furthermore, the functional characterization of the D. melanogaster mitochondrial thiamine pyrophosphate carrier protein (DmTpc1p) is described. DmTpc1p was over-expressed in bacteria, the purified protein was reconstituted into liposomes, and its transport properties and kinetic parameters were characterized. Reconstituted DmTpc1p transports thiamine pyrophosphate and, to a lesser extent, pyrophosphate, ADP, ATP and other nucleotides. The expression of DmTpc1p in Saccharomyces cerevisiaeTPC1 null mutant abolishes the growth defect on fermentable carbon sources. The main role of DmTpc1p is to import thiamine pyrophosphate into mitochondria by exchange with intramitochondrial ATP and/or ADP.


Assuntos
Proteínas de Transporte/metabolismo , Drosophila melanogaster/metabolismo , Mitocôndrias/enzimologia , Tiamina Pirofosfato/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico , Proteínas de Transporte/química , Drosophila melanogaster/genética , Humanos , Cinética , Dados de Sequência Molecular , Mutação/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Tiamina Pirofosfato/química , Tiamina Pirofosfato/genética
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