RESUMO
Pyruvate constitutes a critical branch point in cellular carbon metabolism. We have identified two proteins, Mpc1 and Mpc2, as essential for mitochondrial pyruvate transport in yeast, Drosophila, and humans. Mpc1 and Mpc2 associate to form an ~150-kilodalton complex in the inner mitochondrial membrane. Yeast and Drosophila mutants lacking MPC1 display impaired pyruvate metabolism, with an accumulation of upstream metabolites and a depletion of tricarboxylic acid cycle intermediates. Loss of yeast Mpc1 results in defective mitochondrial pyruvate uptake, and silencing of MPC1 or MPC2 in mammalian cells impairs pyruvate oxidation. A point mutation in MPC1 provides resistance to a known inhibitor of the mitochondrial pyruvate carrier. Human genetic studies of three families with children suffering from lactic acidosis and hyperpyruvatemia revealed a causal locus that mapped to MPC1, changing single amino acids that are conserved throughout eukaryotes. These data demonstrate that Mpc1 and Mpc2 form an essential part of the mitochondrial pyruvate carrier.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Ácido Pirúvico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , Proteínas de Transporte de Ânions/química , Proteínas de Transporte de Ânions/genética , Transporte Biológico , Metabolismo dos Carboidratos , Ciclo do Ácido Cítrico , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/química , Drosophila melanogaster/genética , Humanos , Metabolômica , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Transportadores de Ácidos Monocarboxílicos , Oxirredução , Mutação Puntual , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Small molecule allostery modifies protein function but is not easily discovered. We introduce mass spectrometry integrated with equilibrium dialysis for the discovery of allostery systematically (MIDAS), a method for identifying physiologically relevant, low-affinity metabolite-protein interactions using unmodified proteins and complex mixtures of unmodified metabolites. In a pilot experiment using five proteins, we identified 16 known and 13 novel interactions. The known interactions included substrates, products, intermediates, and allosteric regulators of their protein partners. MIDAS does not depend upon enzymatic measurements, but most of the new interactions affect the enzymatic activity of the protein partner. We found that the fatty acid palmitate interacts with both glucokinase and glycogen phosphorylase. Further characterization revealed that palmitate inhibited both enzymes, possibly providing a mechanism for sparing carbohydrate catabolism when fatty acids are abundant.
Assuntos
Sítio Alostérico , Glicogênio Fosforilase/química , Glicogênio Fosforilase/metabolismo , Metaboloma , Mapeamento de Interação de Proteínas , Proteoma/química , Proteoma/metabolismo , Regulação Alostérica , Animais , Bovinos , Cromatografia Líquida , Cromatografia Gasosa-Espectrometria de Massas , Glucoquinase/química , Glucoquinase/metabolismo , Glutamato Desidrogenase/química , Glutamato Desidrogenase/metabolismo , Humanos , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Projetos Piloto , Saccharomyces cerevisiae/enzimologiaRESUMO
A key factor in the potential clinical utility of membrane-active antibiotics is their cell selectivity (i.e., prokaryote over eukaryote). Cationic steroid antibiotics were developed to mimic the lipid A binding character of polymyxin B and are shown to bind lipid A derivatives with affinity greater than that of polymyxin B. The outer membranes of Gram-negative bacteria are comprised primarily of lipid A, and a fluorophore-appended cationic steroid antibiotic displays very high selectivity for Gram-negative bacterial membranes over Gram-positive bacteria and eukaryotic cell membranes. This cell selectivity of cationic steroid antibiotics may be due, in part, to the affinity of these compounds for lipid A.
Assuntos
Antibacterianos/química , Antibacterianos/farmacocinética , Esteroides/química , Esteroides/farmacocinética , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacocinética , Cátions , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Lipídeo A/metabolismo , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Espectrometria de Fluorescência , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
[reaction: see text] New cationic steroid antibiotics have been prepared by conjugating tripeptides to a triamino analogue of cholic acid. These compounds were synthesized on a solid phase in an indexed library that was screened for antibacterial activity against Gram-negative and Gram-positive bacteria. Selected compounds were synthesized on a larger scale, and minimum inhibition concentrations were measured. These results correlated very well with the screening of the indexed library of antibiotics. The most active antibiotics demonstrate a marked improvement over a related and well characterized cationic steroid antibiotic.
Assuntos
Antibacterianos , Ácidos Cólicos/química , Técnicas de Química Combinatória , Escherichia coli/efeitos dos fármacos , Oligopeptídeos , Staphylococcus aureus/efeitos dos fármacos , Esteroides , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Esteroides/síntese química , Esteroides/química , Esteroides/farmacologiaRESUMO
[reaction: see text] The expedient synthesis of a library of kanamycin B analogues is reported. The revealed SAR will guide future designs in developing kanamycin-type aminoglycoside antibiotics against drug-resistant bacteria.