RESUMEN
Despite the therapeutic importance of antifolates, the links between their direct antimetabolite activity and downstream consequences remain incompletely understood. Here we employ metabolomics to examine the complete metabolic effects of the antibiotic trimethoprim in E. coli. In rich media, trimethoprim treatment causes thymineless death. In minimal media, in contrast, trimethoprim addition results in rapid stoppage of cell growth and stable cell stasis. We show that initial impairment of cell growth is due to rapid depletion of glycine and associated activation of the stringent response. Long-term stasis is due to purine insufficiency. Thus, E. coli has dual systems for surviving folate depletion and avoiding thymineless death: a short-term response based on sensing of amino acids and a long-term response based on sensing of nucleotides.
Asunto(s)
Escherichia coli/citología , Escherichia coli/metabolismo , Glicina/metabolismo , Purinas/metabolismo , Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Antagonistas del Ácido Fólico , Metaboloma , Timina/metabolismo , Trimetoprim/farmacologíaRESUMEN
Mass spectrometry technologies for measurement of cellular metabolism are opening new avenues to explore drug activity. Trimethoprim is an antibiotic that inhibits bacterial dihydrofolate reductase (DHFR). Kinetic flux profiling with (15)N-labeled ammonia in Escherichia coli reveals that trimethoprim leads to blockade not only of DHFR but also of another critical enzyme of folate metabolism: folylpoly-gamma-glutamate synthetase (FP-gamma-GS). Inhibition of FP-gamma-GS is not directly due to trimethoprim. Instead, it arises from accumulation of DHFR's substrate dihydrofolate, which we show is a potent FP-gamma-GS inhibitor. Thus, owing to the inherent connectivity of the metabolic network, falling DHFR activity leads to falling FP-gamma-GS activity in a domino-like cascade. This cascade results in complex folate dynamics, and its incorporation in a computational model of folate metabolism recapitulates the dynamics observed experimentally. These results highlight the potential for quantitative analysis of cellular metabolism to reveal mechanisms of drug action.
Asunto(s)
Proteínas de Escherichia coli/antagonistas & inhibidores , Escherichia coli/efectos de los fármacos , Antagonistas del Ácido Fólico/farmacología , Complejos Multienzimáticos/antagonistas & inhibidores , Péptido Sintasas/antagonistas & inhibidores , Trimetoprim/farmacología , Simulación por Computador , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Complejos Multienzimáticos/metabolismo , Péptido Sintasas/metabolismo , Tetrahidrofolato Deshidrogenasa/efectos de los fármacos , Tetrahidrofolato Deshidrogenasa/metabolismoRESUMEN
Folate metabolism, which is responsible for one-carbon transfer reactions in critical cellular processes including thymidine biosynthesis, is among the most important targets of antibiotic and anticancer drugs. Analysis of intracellular folates is complicated by three different types of folate modification: oxidation/reduction, methylation, and polyglutamylation. Here we present a method for quantifying the full diversity of intracellular folates by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method begins with folate extraction using -75 degrees C methanol:water, with ascorbic acid and ammonium acetate added to prevent folate interconversion. The extract is then separated using hydrophilic interaction chromatography with an amino column, ionized by positive mode electrospray, and analyzed on a triple quadrupole instrument using multiple reaction monitoring. The method has been used to profile the folate pools in Escherichia coli and Saccharomyces cerevisiae, with absolute levels of selected folates in E. coli measured by spiking extracts of cells fed uniformly (13)C-glucose with purified, unlabeled folate standards. An isotope-ratio-based approach has been applied to study the effects of trimethoprim, a clinically important antibiotic that blocks bacterial dihydrofolate reductase. In addition to causing the expected increase in oxidized and decrease in reduced folates, trimethoprim triggered a dramatic and previously unrecognized shift towards shorter polyglutamate chain lengths. This finding highlights the potential for analysis of the full spectrum of cellular folates by MS/MS to unveil novel biological phenomena.
Asunto(s)
Isótopos de Carbono/química , Escherichia coli/química , Ácido Fólico/química , Saccharomyces cerevisiae/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Cromatografía Líquida de Alta Presión , Escherichia coli/metabolismo , Ácido Fólico/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
Autophagic cell death is characterized by the accumulation of vacuoles in physiological and pathological conditions. However, its molecular event is unknown. Here, we show that Atg5, which is known to function in autophagy, contributes to autophagic cell death by interacting with Fas-associated protein with death domain (FADD). Down-regulation of Atg5 expression in HeLa cells suppresses cell death and vacuole formation induced by IFN-gamma. Inversely, ectopic expression of Atg5 using adenoviral delivery induces autophagic cell death. Deletion mapping analysis indicates that procell death activity resides in the middle and C-terminal region of Atg5. Cells harboring the accumulated vacuoles triggered by IFN-gamma or Atg5 expression become dead, and vacuole formation precedes cell death. 3-Methyladenine or expression of Atg5(K130R) mutant blocks both cell death and vacuole formation triggered by IFN-gamma, whereas benzyloxycarbonyl-VAD-fluoromethyl ketone (Z-VAD-fmk) inhibits only cell death but not vacuole formation. Atg5 interacts with FADD via death domain in vitro and in vivo, and the Atg5-mediated cell death, but not vacuole formation, is blocked in FADD-deficient cells. These results suggest that Atg5 plays a crucial role in IFN-gamma-induced autophagic cell death by interacting with FADD.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Adenina/análogos & derivados , Autofagia/fisiología , Muerte Celular/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , Vacuolas/fisiología , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Adenina/farmacología , Clorometilcetonas de Aminoácidos/farmacología , Proteína 5 Relacionada con la Autofagia , Sitios de Unión , Muerte Celular/efectos de los fármacos , Línea Celular , Proteína de Dominio de Muerte Asociada a Fas , Eliminación de Gen , Expresión Génica , Células HeLa , Humanos , Interferón gamma/farmacología , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Mutagénesis , Oligonucleótidos Antisentido/farmacología , Saccharomyces cerevisiae , Transfección , Técnicas del Sistema de Dos Híbridos , Vacuolas/efectos de los fármacosRESUMEN
In response to a diverse array of signals, IkappaBalpha is targeted for phosphorylation-dependent degradation by the proteasome, thereby activating NF-kappaB. Here we demonstrate a role of the cleavage product of IkappaBalpha in various death signals. During apoptosis of NIH3T3, Jurkat, Rat-1, and L929 cells exposed to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), Fas, serum deprivation, or TNF-alpha, respectively, IkappaBalpha was cleaved in a caspase-dependent manner. In vitro and in vivo cleavage assays and site-directed mutagenesis showed that caspase-3 cleaved IkappaBalpha between Asp31 and Ser32. Expression of the cleavage product lacking amino-terminus (1-31), DeltaIkappaBalpha, sensitized otherwise resistant NIH3T3 fibroblast cells to apoptosis induced by TNF-alpha or TRAIL, and HeLa tumor cells to TNF-alpha. DeltaIkappaBalpha was more pro-apoptotic compared to wild type or cleavage-resistant (D31E)IkappaBalpha mutant and the sensitization elicited by DeltaIkappaBalpha was as effective as that by the dominant negative mutant, (S32,36A)IkappaBalpha, in NIH3T3 cells. DeltaIkappaBalpha suppressed the transactivation of NF-kappaB induced by TNF-alpha or TRAIL, as reflected by luciferase-reporter activity. Conversely, expression of the p65 subunit of NF-kappaB suppressed TNF-alpha-, TRAIL-, and serum deprivation-induced cell death. On the contrary, DeltaIkappaBalpha was less effective at increasing the death rate of HeLa cells that were already sensitive to death signals including TRAIL, etoposide, or taxol. These results suggest that DeltaIkappaBalpha generated by various death signals sensitizes cells to apoptosis by suppressing NF-kappaB activity.