RESUMO
All medically useful antibiotics should have the potential to distinguish between target microbes (bacteria) and host cells. Although many antibiotics that target bacterial protein synthesis show little effect on the translation machinery of the eukaryotic cytoplasm, it is unclear whether these antibiotics target or not the mitochondrial translation machinery. We employed an in vitro translation system from bovine mitochondria, which consists of mitochondrial ribosomes and mitochondrial elongation factors, to estimate the effect of antibiotics on mitichondrial protein synthesis. Tetracycline and thiostrepton showed similar inhibitory effects on both Escherichia coli and mitochondrial protein synthesis. The mitochondrial system was more resistant to tiamulin, macrolides, virginiamycin, fusidic acid and kirromycin than the E. coli system. The present results, taken together with atomic structure of the ribosome, may provide useful information for the rational design of new antibiotics having less adverse effects in humans and animals.
Assuntos
Antibacterianos/farmacologia , Mitocôndrias/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Animais , Bovinos , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Mitocôndrias/metabolismo , Fatores de Alongamento de Peptídeos/efeitos dos fármacos , Ribossomos/efeitos dos fármacosRESUMO
A remarkable positive cooperativity between the GTPase activities of EF-Tu and EF-G on empty ribosomes from Escherichia coli has been discovered. This cooperativity implies a decrease of the corresponding apparent KM values of the empty ribosome for either elongation factor: from more than 10 microM to 0.5 microM for EF-Tu.GTP by the addition of 0.25 microM EF-G and from 0.7 microM to 0.5 microM for EF-G.GTP by the addition of 3 microM EF-Tu. In a further analysis of this phenomenon, the effects of various specific antibiotics were studied: thiostrepton, fusidic acid, tetracycline, pulvomycin and kirromycin appeared to inhibit the synergistic effect, whereas streptomycin was found to stimulate it. Even in the present minimal system the ribosomes respond to the above-mentioned antibiotics in a way surprisingly similar to that in the coupled system with mRNA and tRNAs. The cooperativity seems not to be due to a simultaneous binding of the two elongation factors to the ribosome as revealed by studying the effects of fusidic acid and kirromycin, and by band-shift experiments by means of gel electrophoresis under non-denaturing conditions. Our experimental data and the kinetic analysis of alternative models provide evidence that EF-Tu.GTP and EF-G.GTP interact sequentially with empty ribosomes that oscillate between two different conformations, one for each elongation factor. Apparently, ribosomes have an intrinsic property for oscillation as normally observed during protein synthesis with a frequency paced by the events of tRNA binding and translocation.
Assuntos
Fatores de Elongação Ligados a GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Fator Tu de Elongação de Peptídeos/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Ribossomos/metabolismo , Regulação Alostérica/fisiologia , Antibacterianos/farmacologia , Eletroforese , Escherichia coli/metabolismo , Fatores de Elongação Ligados a GTP Fosfo-Hidrolases/efeitos dos fármacos , Cinética , Modelos Biológicos , Fator G para Elongação de Peptídeos , Fator Tu de Elongação de Peptídeos/efeitos dos fármacos , Fatores de Alongamento de Peptídeos/efeitos dos fármacos , Ligação Proteica , Piridonas/farmacologia , Estreptomicina/farmacologiaRESUMO
An appropriate mixture of ethylene glycol and BaCl2 enhanced the otherwise very low intrinsic GTPase activity of the elongation factor 2 isolated from the archaeon Sulfolobus solfataricus (SsEF-2). The enzymatic activity became up to 300-fold higher than that of the SsEF-2 GTPase measured in the absence of any stimulator, but remained 20-fold lower than that stimulated by ribosome. The stimulatory effect of ethylene glycol/Ba2+ was attributed to the increased affinity for GTP, probably related to a conformational change occurring in a hydrophobic region near the catalytic site.
Assuntos
Álcoois/farmacologia , Guanosina Trifosfato/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Sulfolobus/enzimologia , Adenosina Difosfato Ribose , Compostos de Bário/farmacologia , Sítios de Ligação , Cátions Bivalentes/farmacologia , Cloretos/farmacologia , Etilenoglicol , Etilenoglicóis/farmacologia , Fatores de Elongação Ligados a GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Guanosina Difosfato/farmacologia , Guanosina Trifosfato/farmacologia , Guanilil Imidodifosfato/metabolismo , Guanilil Imidodifosfato/farmacologia , Concentração de Íons de Hidrogênio , Hidrólise , Fator 2 de Elongação de Peptídeos , Fatores de Alongamento de Peptídeos/efeitos dos fármacos , TemperaturaRESUMO
Bolesatine is a potent cytotoxic glycoprotein purified from Boletus satanas Lenz, which has previously been shown to be an inhibitor of protein synthesis in several in vitro systems and in vivo. For a better understanding of its mechanism of action on protein synthesis at the ribosomal level, rat liver ribosomes were pretreated with bolesatine (1 to 10 micrograms) added to in vitro polyuridylic acid (poly(U)) translation systems before and after washing. The fact that ribosomes were still active confirmed that bolesatine cannot be included in the group of protein synthesis inhibitors of plant origin, known as ribosome-inactivating proteins (RIPs). The effect of bolesatine on the EF-2 elongation factor and post-ribosomal fraction was then studied in vitro. The results indicated that bolesatine does not have a direct effect on elongation factors, but hydrolyses the nucleoside triphosphates, GTP (80% to 90%, respectively for 1 to 10 micrograms) and ATP (10% to 40%, respectively for 1 to 10 micrograms), with consequent inhibition of protein synthesis. Thus, bolesatine should be classified as a nucleoside triphosphate phosphatase, rather than as a direct inhibitor of protein synthesis. The study of the effect of bolesatine on the EF-2 factor revealed that the mechanism whereby bolesatine affects protein synthesis probably involves GTP hydrolysis rather than EF-2 inhibition.
Assuntos
Proteínas Fúngicas/toxicidade , Fígado/efeitos dos fármacos , Micotoxinas/toxicidade , Inibidores da Síntese de Proteínas/toxicidade , Trifosfato de Adenosina/metabolismo , Animais , Basidiomycota , Proteínas Fúngicas/administração & dosagem , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/farmacocinética , Guanosina Trifosfato/metabolismo , Hidrólise , Técnicas In Vitro , Micotoxinas/administração & dosagem , Micotoxinas/isolamento & purificação , Micotoxinas/farmacocinética , Proteínas do Tecido Nervoso/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Fator 2 de Elongação de Peptídeos , Fatores de Alongamento de Peptídeos/efeitos dos fármacos , Fatores de Alongamento de Peptídeos/metabolismo , Fenilalanina/química , Fenilalanina/metabolismo , Poli U/farmacologia , Ratos , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismoRESUMO
Various species of Taxus contain taxanes that promote polymerization and stabilization of microtubules. They have been reported as antineoplastic compounds with highly effective chemiotherapeutic application. A decrease in incorporation of the radiolabelled precursors into DNA, RNA and proteins in vivo has been reported too. The preliminary results have shown that also the other compounds present in the aqueous extract from Taxus baccata needles, participate in the inhibition of the protein biosynthesis. The binding site of eEF-2 on the ribosome seems to be the target of this inhibition process.
Assuntos
Fígado/efeitos dos fármacos , Paclitaxel/farmacologia , Fatores de Alongamento de Peptídeos/efeitos dos fármacos , Adrenérgicos/farmacologia , Animais , Sistema Livre de Células , Efedrina/farmacologia , Fígado/metabolismo , Masculino , Fatores de Alongamento de Peptídeos/biossíntese , Extratos Vegetais/farmacologia , Ratos , Ratos WistarRESUMO
Translation elongation factor 2 (EF2), which in Saccharomyces cerevisiae is expressed from the EFT1 and EFT2 genes, has been found to be targeted by a new family of highly specific antifungal compounds derived from the natural product sordarin. Two complementation groups of mutants resistant to the semisynthetic sordarin derivative GM193663 were found. The major one (21 members) consisted of isolates with mutations on EFT2. The minor one (four isolates) is currently being characterized but it is already known that resistance in this group is not due to mutations on EFT1, pointing to the complex structure of the functional target for these compounds. Mutations on EF2 clustered, forming a possible drug binding pocket on a three-dimensional model of EF2, and mutant cell extracts lost the capacity to bind to the inhibitors. This new family of antifungals holds the promise to be a much needed and potent addition to current antimicrobial treatments, as well as a useful tool for dissection of the elongation process in ribosomal protein synthesis.
Assuntos
Antifúngicos/farmacologia , Fatores de Alongamento de Peptídeos/efeitos dos fármacos , Sequência de Aminoácidos , Sítios de Ligação , Resistência Microbiana a Medicamentos , Indenos , Dados de Sequência Molecular , Mutação , Fator 2 de Elongação de Peptídeos , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/genéticaRESUMO
We have used protein engineering and recombinant DNA methodologies in order to construct a fusion protein in which human interleukin-2 (IL-2) is genetically linked to the catalytic and transmembrane domains of diphtheria toxin. The fusion toxin, DAB486IL-2, is highly cytotoxic for only those cells which display the high affinity interleukin-2 receptor (IL-2R) on their surface. In phase I/II clinical studies the intravenous administration of DAB486IL-2 has been found to be safe, well tolerated and may lead to the induction of durable remissions in patients presenting with a variety of IL-2R positive lymphomas.
Assuntos
Antineoplásicos/farmacologia , Toxina Diftérica/química , Fatores de Alongamento de Peptídeos/efeitos dos fármacos , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/farmacologia , Catálise , Humanos , Modelos Moleculares , Fator 2 de Elongação de Peptídeos , Estrutura Terciária de ProteínaRESUMO
GR135402, a sordarin derivative, was isolated in an antifungal screening program. GR135402, sordarin, and derivatives of both compounds were evaluated for their ability to inhibit cell-free translational systems from five different pathogenic fungi (Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, and Cryptococcus neoformans). The activity profile of GR135402 is extended to other chemical compounds derived from sordarin. Experimental results indicate that sordarin analogs exert their antifungal effects by specifically inhibiting the protein synthesis elongation cycle in yeasts but do not affect protein synthesis machinery in mammalian systems. Intrinsically resistant strains owe their resistance to differences in the molecular target of sordarins in these strains. Preliminary studies performed to elucidate the mode of action of this new class of antifungal agents have shown that the putative target of sordarins is one of the protein synthesis elongation factors.
Assuntos
Antifúngicos/farmacologia , Proteínas Fúngicas/biossíntese , Fatores de Alongamento de Peptídeos/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Candida/efeitos dos fármacos , Indenos , RNA Fúngico/biossínteseRESUMO
We have studied the effect of oxidative stress on protein synthesis in rat liver. Cumene hydroperoxide (CH) was used as an oxidant agent. The approach used was to determine the ribosomal state of aggregation and the time for assembly and release of polypeptide chains in the process of protein synthesis in rat liver in vivo. The results suggest that the elongation step is the most sensitive to CH treatment. The measurement of both carbonyl groups content and ADP-ribosylatable elongation factor 2 (EF-2), the main protein involved in the elongation step, indicates that under CH treatment EF-2 is oxidatively modified and a lower amount of active EF-2 is present. These results are corroborated by in vitro oxidation of EF-2 and could explain for the decline in the elongation step.
Assuntos
Derivados de Benzeno/farmacologia , Fígado/metabolismo , Oxidantes/farmacologia , Estresse Oxidativo/fisiologia , Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , Fatores de Alongamento de Peptídeos/efeitos dos fármacos , Adenosina Difosfato Ribose/metabolismo , Animais , Feminino , Malondialdeído/análise , Oxirredução , Fator 2 de Elongação de Peptídeos , Polirribossomos/metabolismo , Ratos , Ratos WistarRESUMO
The target for sordarins in Candida albicans has been elucidated. Kinetic experiments of sordarin inhibition as well as displacement experiments showed that the formation of a sordarin-target complex follows a reversible mechanism. Binding of tritiated drug to the target is enhanced in the presence of ribosomes. Isolation of the target by classical protein purification methods has allowed us to identify it as elongation factor 2. This is in agreement with the nature of sordarin derivatives as specific inhibitors of the elongation cycle within protein synthesis in yeasts.
Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Fatores de Alongamento de Peptídeos/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Sequência de Aminoácidos , Candida albicans/metabolismo , Proteínas de Transporte/isolamento & purificação , Indenos , Dados de Sequência Molecular , Fator 2 de Elongação de PeptídeosRESUMO
To examine the role of phosphorylation of the elongation factor eEF-1 in regulation of translation, 32P-labeled 3T3-L1 cells were deprived of serum, then incubated in the presence or absence of 10 nM insulin for 15 min. eEF-1 was purified by affinity chromatography on tRNA-Sepharose and shown to be phosphorylated on the alpha, beta and delta subunits. Phosphorylation of eEF-1alpha was stimulated sixfold in response to insulin, beta was stimulated fourfold and delta was threefold. The rate of elongation assayed with eEF-1 from insulin-stimulated cells was over twofold greater than with eEF-1 from serum-deprived cells. When eEF-1 from insulin-treated cells was subjected to two-dimensional tryptic phosphopeptide mapping, nine phosphopeptides were obtained with the alpha subunit, one with the beta subunit and three with the delta subunit. When compared with phosphopeptide maps of alpha, beta and delta subunits of eEF-1 phosphorylated in vitro by the insulin-stimulated multipotential protein kinase, the maps of the beta and delta subunits were identical. Five phosphopeptides obtained with the alpha subunit in vivo were identical to those obtained with S6 kinase in vitro; the remainder were unique. To examine whether protein kinase C had a role in phosphorylation of eEF-1 in response to insulin, protein kinase C was down-regulated by prolonged exposure of 3T3-L1 cells to 4beta-phorbol 12-myristate 13-acetate (PMA). Phosphorylation of the alpha, beta and delta subunits was stimulated 2.5-fold in response to insulin, with elongation activity stimulated to a similar extent, suggesting that protein kinase C had no effect on stimulation of elongation in response to insulin. Thus, stimulation of eEF-1 activity in response to insulin appears to be mediated primarily by multipotential S6 kinase. This data is consistent with previous studies on stimulation of initiation via phosphorylation of initiation factors by multipotential S6 kinase [Morley, S. J. & Traugh, J. A. (1993) Biochemie (Paris) 95, 985-989].
Assuntos
Insulina/farmacologia , Fatores de Alongamento de Peptídeos/metabolismo , Células 3T3/efeitos dos fármacos , Células 3T3/metabolismo , Animais , Meios de Cultura Livres de Soro , Regulação para Baixo , Camundongos , Fator 1 de Elongação de Peptídeos , Fatores de Alongamento de Peptídeos/efeitos dos fármacos , Fosfoproteínas/análise , Fosfoproteínas/química , Fosforilação/efeitos dos fármacos , Coelhos , Proteínas Quinases S6 Ribossômicas/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Sordarin derivatives are selective inhibitors of fungal protein synthesis, which specifically impair elongation factor 2 (EF-2) function. We have studied the effect of sordarin on the ribosome-dependent GTPase activity of EF-2 from Candida albicans in the absence of any other component of the translation system. The effect of sordarin turned out to be dependent both on the ratio of ribosomes to EF-2 and on the nature of the ribosomes. When the amount of EF-2 exceeded that of ribosomes sordarin inhibited the GTPase activity following an inverted bell-shaped dose-response curve, whereas when EF-2 and ribosomes were in equimolar concentrations sordarin yielded a typical sigmoidal dose-dependent inhibition. However, when ricin-treated ribosomes were used, sordarin stimulated the hydrolysis of GTP. These results were compared with those obtained with fusidic acid, showing that both drugs act in a different manner. All these data are consistent with sordarin blocking the elongation cycle at the initial steps of translocation, prior to GTP hydrolysis. In agreement with this conclusion, sordarin prevented the formation of peptidyl-[(3)H]puromycin on polysomes from Candida albicans.
Assuntos
Antifúngicos/farmacologia , Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Candida albicans , Relação Dose-Resposta a Droga , Ácido Fusídico/farmacologia , Fatores de Elongação Ligados a GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Hidrólise , Indenos , Modelos Biológicos , Fator 2 de Elongação de Peptídeos , Fatores de Alongamento de Peptídeos/efeitos dos fármacos , Puromicina/metabolismo , Ribossomos/efeitos dos fármacos , Ricina/farmacologiaRESUMO
The elongation factor complex, EF-1H, serves an essential function in protein biosynthesis in eukaryotic cells, although the role of EF-1H in other physiological processes is unknown. In this report, we demonstrate that three components of EF-1H (EF-1 beta, EF-1 delta, EF-1 gamma) bind to DNA modified with chromium (Cr), a potent DNA-damaging agent and an established human carcinogen. The EF-1H complex also binds to transplatin modified DNA but not to cisplatin-modified DNA. These results demonstrate that the EF-1H complex has functional DNA binding activity and is capable of recognizing the distortions in DNA structure resulting from the covalent binding of Cr and transplatin to DNA.
Assuntos
Cromo/toxicidade , Cisplatino/toxicidade , Adutos de DNA , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Carcinoma , Reagentes de Ligações Cruzadas/toxicidade , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/isolamento & purificação , Feminino , Humanos , Ligantes , Dados de Sequência Molecular , Proteínas de Neoplasias/efeitos dos fármacos , Proteínas de Neoplasias/isolamento & purificação , Proteínas de Neoplasias/metabolismo , Neoplasias Ovarianas , Fatores de Alongamento de Peptídeos/efeitos dos fármacos , Fatores de Alongamento de Peptídeos/isolamento & purificação , Células Tumorais CultivadasRESUMO
The oxazolidinones are a novel class of antimicrobial agents that target protein synthesis in a wide spectrum of gram-positive and anaerobic bacteria. The oxazolidinone PNU-100766 (linezolid) inhibits the binding of fMet-tRNA to 70S ribosomes. Mutations to oxazolidinone resistance in Halobacterium halobium, Staphylococcus aureus, and Escherichia coli map at or near domain V of the 23S rRNA, suggesting that the oxazolidinones may target the peptidyl transferase region responsible for binding fMet-tRNA. This study demonstrates that the potency of oxazolidinones corresponds to increased inhibition of fMet-tRNA binding. The inhibition of fMet-tRNA binding is competitive with respect to the fMet-tRNA concentration, suggesting that the P site is affected. The fMet-tRNA reacts with puromycin to form peptide bonds in the presence of elongation factor P (EF-P), which is needed for optimum specificity and efficiency of peptide bond synthesis. Oxazolidinone inhibition of the P site was evaluated by first binding fMet-tRNA to the A site, followed by translocation to the P site with EF-G. All three of the oxazolidinones used in this study inhibited translocation of fMet-tRNA. We propose that the oxazolidinones target the ribosomal P site and pleiotropically affect fMet-tRNA binding, EF-P stimulated synthesis of peptide bonds, and, most markedly, EF-G-mediated translocation of fMet-tRNA into the P site.