RESUMEN
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is considered a classical glycolytic protein that can promote the fusion of phospholipid vesicles and can also play a vital role on in vivo fusogenic events. However, it is not clear how this redox enzyme, which lack conserved structural or sequence motifs related to membrane fusion, catalyze this process. In order to detect if this ability is present in other NAD(P)H dehydrogenases with available structure, spectroscopic studies were performed to evaluate the capability of alcohol dehydrogenase (ADH), glutamic dehydrogenase (GDH) and sorbitol dehydrogenase (SDH) to bind, aggregate, destabilize and fuse vesicles. Based on finite difference Poisson-Boltzmann calculations (FDPB) the protein-membrane interactions were analyzed. A model for the protein-membrane complex in its minimum free energy of interaction was obtained for each protein and the amino acids involved in the binding processes were suggested. A previously undescribed relationship between membrane destabilization and crevices with high electropositive potential on the protein surface was proposed. The putative implication of the non-specific electrostatics on NAD(P)H dehydrogenases induced membrane fusion is discussed.
Asunto(s)
Fusión de Membrana , NADH NADPH Oxidorreductasas/química , Liposomas Unilamelares/química , Alcohol Deshidrogenasa/química , Animales , Secuencia de Bases , Bovinos , Secuencia Conservada , Glutamato Deshidrogenasa (NADP+)/química , Gliceraldehído 3-Fosfato Deshidrogenasa (NADP+)/química , L-Iditol 2-Deshidrogenasa/química , Modelos Moleculares , Fosfatidilcolinas/química , Fosfatidilserinas/química , Estructura Secundaria de Proteína , Conejos , Ovinos , Espectrometría de Fluorescencia , Electricidad Estática , TermodinámicaRESUMEN
Microcin J25 (MccJ25) uptake by Escherichia coli requires the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbD, ExbB, and SbmA. MccJ25 appears to have two intracellular targets: (i) RNA polymerase (RNAP), which has been described in E. coli and Salmonella enterica serovars, and (ii) the respiratory chain, reported only in S. enterica serovars. In the current study, it is shown that the observed difference between the actions of microcin on the respiratory chain in E. coli and S. enterica is due to the relatively low microcin uptake via the chromosomally encoded FhuA. Higher expression by a plasmid-encoded FhuA allowed greater uptake of MccJ25 by E. coli strains and the consequent inhibition of oxygen consumption. The two mechanisms, inhibition of RNAP and oxygen consumption, are independent of each other. Further analysis revealed for the first time that MccJ25 stimulates the production of reactive oxygen species (O(2)(*-)) in bacterial cells, which could be the main reason for the damage produced on the membrane respiratory chain.
Asunto(s)
Bacteriocinas/farmacología , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efectos de los fármacos , Superóxidos/metabolismo , Antibacterianos/farmacocinética , Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Bacteriocinas/farmacocinética , Catalasa/genética , Catalasa/metabolismo , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , ARN Polimerasas Dirigidas por ADN/genética , Activación Enzimática/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/genética , Genotipo , Consumo de Oxígeno/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Microcin J25 appears to have two intracellular targets: (1) RNA polymerase, which was described in Escherichia coli and Salmonella enterica serovars, and (2) cell respiration in Salmonella enterica serovars. C-terminal glycine amidation of the threaded segment localized in the MccJ25 lariat ring region specifically blocked the RNA-polymerase inhibition, but not the cell respiration inhibition and peptide uptake. These results suggest that different regions of the molecule are responsible for each cellular effect, they are localized far away from the beta-hairpin region and the C-terminal region is an important determinant for RNAP inhibition.
Asunto(s)
Bacteriocinas/química , Bacteriocinas/metabolismo , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Amidas/metabolismo , Bacteriocinas/genética , Respiración de la Célula/fisiología , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/clasificación , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Consumo de Oxígeno , ARN/biosíntesis , Salmonella/clasificación , Salmonella/genética , Salmonella/crecimiento & desarrollo , Salmonella/metabolismo , Transcripción GenéticaRESUMEN
The antibiotic microcin J25 (MccJ25) was cleaved by hydrolysis with thermolysin giving a two-chain peptide (MccJ25-Th19) of 10 and 9 amino acid residues. MccJ25-Th19 with deep modifications in beta-hairpin region had no effect on Escherichia coli growth, but still inhibited RNA polymerase in vitro and oxygen consumption in Salmonella strains. MccJ25-Th19 showed antibiotic activity on E. coli transformed with plasmids containing either fhuA or sbmA genes, which code for proteins involved in MccJ25 transport. These results suggest that an intact beta-hairpin region is crucial for MccJ25 import but not for inhibition of E. coli RNA polymerase or oxygen consumption in Salmonella strains.