RESUMEN
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.
Asunto(s)
Proteínas de Transporte de Anión/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Ácido Pirúvico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Animales , Proteínas de Transporte de Anión/química , Proteínas de Transporte de Anión/genética , Transporte Biológico , Metabolismo de los Hidratos de Carbono , Ciclo del Ácido Cítrico , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/química , Drosophila melanogaster/genética , Humanos , Metabolómica , Proteínas de Transporte de Membrana Mitocondrial/química , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Datos de Secuencia Molecular , Transportadores de Ácidos Monocarboxílicos , Oxidación-Reducción , Mutación Puntual , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
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.
Asunto(s)
Sitio Alostérico , Glucógeno Fosforilasa/química , Glucógeno Fosforilasa/metabolismo , Metaboloma , Mapeo de Interacción de Proteínas , Proteoma/química , Proteoma/metabolismo , Regulación Alostérica , Animales , Bovinos , Cromatografía Liquida , Cromatografía de Gases y Espectrometría de Masas , Glucoquinasa/química , Glucoquinasa/metabolismo , Glutamato Deshidrogenasa/química , Glutamato Deshidrogenasa/metabolismo , Humanos , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proyectos Piloto , Saccharomyces cerevisiae/enzimologíaRESUMEN
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.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacocinética , Esteroides/química , Esteroides/farmacocinética , Materiales Biomiméticos/química , Materiales Biomiméticos/farmacocinética , Cationes , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Lípido A/metabolismo , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/metabolismo , Espectrometría de Fluorescencia , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/metabolismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
[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.
Asunto(s)
Antibacterianos , Ácidos Cólicos/química , Técnicas Químicas Combinatorias , Escherichia coli/efectos de los fármacos , Oligopéptidos , Staphylococcus aureus/efectos de los fármacos , Esteroides , Antibacterianos/síntesis química , Antibacterianos/química , Antibacterianos/farmacología , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Oligopéptidos/síntesis química , Oligopéptidos/química , Oligopéptidos/farmacología , Esteroides/síntesis química , Esteroides/química , Esteroides/farmacologíaRESUMEN
[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.