RESUMEN
Myo-inositol is a precursor of several membrane phospholipids and sphingolipids and plays a key role in gene regulation in Saccharomyces cerevisiae (S. cerevisiae). Here, we tested whether H2O2 was affecting the levels of the inositol transporters and thus inositol uptake. In S. cerevisiae cells adapted to H2O2 Itr1-GFPp accumulated in the plasma membrane until 20 min, concomitantly with an inhibition of its internalization. Exposure to H2O2 did not alter Itr2-GFPp cellular levels and induced only an 8% decrease at 10 min in the plasma membrane. Therefore, decreased inositol intracellular levels are not caused by decreased levels of inositol transporters in the plasma membrane. However, results show that H2O2 adaptation affects Itr1p turnover and, consequently, H2O2-adapted yeast cells display an inositol transporter phenotype comparable to cells grown in the absence of inositol in growth medium, i.e. accumulation in the plasma membrane and decreased degradation.
Asunto(s)
Peróxido de Hidrógeno/farmacología , Inositol/metabolismo , Proteínas de Transporte de Membrana/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Adaptación Fisiológica , Transporte Biológico/efectos de los fármacos , Transporte Biológico/genética , Proteínas de Transporte de Membrana/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
During exposure of yeast cells to low levels of hydrogen peroxide (H2 O2 ), the expression of several genes is regulated for cells to adapt to the surrounding oxidative environment. Such adaptation involves modification of plasma membrane lipid composition, reorganization of ergosterol-rich microdomains and altered gene expression of proteins involved in lipid and vesicle traffic, to decrease permeability to exogenous H2 O2 . Opi1p is a transcriptional repressor that is inactive when present at the nuclear membrane/endoplasmic reticulum, but represseses transcription of inositol upstream activating sequence (UASINO )-containing genes, many of which are involved in the synthesis of phospholipids and fatty acids, when it is translocated to the nucleus. We investigated whether H2 O2 in concentrations inducing adaptation regulates Opi1p function. We found that, in the presence of H2 O2 , GFP-Opi1p fusion protein translocates to the nucleus and, concomitantly, the expression of UASINO -containing genes is affected. We also investigated whether cysteine residues of Opi1p were implicated in the H2 O2 -mediated translocation of this protein to the nucleus and identified cysteine residue 159 as essential for this process. Our work shows that Opi1p is redox-regulated and establishes a new mechanism of gene regulation involving Opi1p, which is important for adaptation to H2 O2 in yeast cells. Copyright © 2017 John Wiley & Sons, Ltd.
Asunto(s)
Núcleo Celular/metabolismo , Retículo Endoplásmico/metabolismo , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Proteínas Represoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Adaptación Biológica , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/efectos de los fármacos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Ácidos Grasos/biosíntesis , Peróxido de Hidrógeno/química , Concentración de Iones de Hidrógeno , Inositol/análisis , Inositol/química , Microdominios de Membrana/metabolismo , Proteínas de Transporte de Monosacáridos/efectos de los fármacos , Proteínas de Transporte de Monosacáridos/genética , Mio-Inositol-1-Fosfato Sintasa/efectos de los fármacos , Mio-Inositol-1-Fosfato Sintasa/genética , Oxidación-Reducción , Estrés Oxidativo , Permeabilidad , Fosfolípidos/biosíntesis , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/efectos de los fármacosRESUMEN
The regulatory mechanisms by which hydrogen peroxide (H2O2) modulates the activity of transcription factors in bacteria (OxyR and PerR), lower eukaryotes (Yap1, Maf1, Hsf1 and Msn2/4) and mammalian cells (AP-1, NRF2, CREB, HSF1, HIF-1, TP53, NF-κB, NOTCH, SP1 and SCREB-1) are reviewed. The complexity of regulatory networks increases throughout the phylogenetic tree, reaching a high level of complexity in mammalians. Multiple H2O2 sensors and pathways are triggered converging in the regulation of transcription factors at several levels: (1) synthesis of the transcription factor by upregulating transcription or increasing both mRNA stability and translation; (ii) stability of the transcription factor by decreasing its association with the ubiquitin E3 ligase complex or by inhibiting this complex; (iii) cytoplasm-nuclear traffic by exposing/masking nuclear localization signals, or by releasing the transcription factor from partners or from membrane anchors; and (iv) DNA binding and nuclear transactivation by modulating transcription factor affinity towards DNA, co-activators or repressors, and by targeting specific regions of chromatin to activate individual genes. We also discuss how H2O2 biological specificity results from diverse thiol protein sensors, with different reactivity of their sulfhydryl groups towards H2O2, being activated by different concentrations and times of exposure to H2O2. The specific regulation of local H2O2 concentrations is also crucial and results from H2O2 localized production and removal controlled by signals. Finally, we formulate equations to extract from typical experiments quantitative data concerning H2O2 reactivity with sensor molecules. Rate constants of 140 M(-1) s(-1) and ≥1.3 × 10(3) M(-1) s(-1) were estimated, respectively, for the reaction of H2O2 with KEAP1 and with an unknown target that mediates NRF2 protein synthesis. In conclusion, the multitude of H2O2 targets and mechanisms provides an opportunity for highly specific effects on gene regulation that depend on the cell type and on signals received from the cellular microenvironment.
Asunto(s)
Proteínas Bacterianas/fisiología , Regulación de la Expresión Génica/fisiología , Peróxido de Hidrógeno/metabolismo , Proteínas Represoras/fisiología , Transducción de Señal/fisiología , Factores de Transcripción/fisiología , Transcripción Genética/fisiología , Animales , Compartimento Celular , Cisteína/metabolismo , Proteínas Fúngicas/fisiología , Regulación Bacteriana de la Expresión Génica/fisiología , Regulación Fúngica de la Expresión Génica/fisiología , Humanos , Oxidación-Reducción , Oxidorreductasas/metabolismo , Estabilidad Proteica , Estabilidad del ARN , Activación TranscripcionalRESUMEN
Hydrogen peroxide (H2O2) at moderate steady-state concentrations synergizes with TNF-α, leading to increased nuclear levels of NF-κB p65 subunit and to a cell-type specific up-regulation of a limited number of NF-κB-dependent genes. Here, we address how H2O2 achieves this molecular specificity. HeLa and MCF-7 cells were exposed to steady-state H2O2 and/or TNF-α and levels of c-Rel, p65, IκB-α, IκB-ß and IκB-ε were determined. For an extracellular concentration of 25 µM H2O2, the intracellular H2O2 concentration is 3.7 µM and 12.5 µM for respectively HeLa and MCF-7 cells. The higher cytosolic H2O2 concentration present in MCF-7 cells may be a contributing factor for the higher activation of NF-κB caused by H2O2 in this cell line, when compared to HeLa cells. In both cells lines, H2O2 precludes the recovery of TNF-α-dependent IκB-α degradation, which may explain the observed synergism between H2O2 and TNF-α concerning p65 nuclear translocation. In MCF-7 cells, H2O2, in the presence of TNF-α, tripled the induction of c-Rel triggered either by TNF-α or H2O2. Conversely, in HeLa cells, H2O2 had a small antagonistic effect on TNF-α-induced c-Rel nuclear levels, concomitantly with a 50 % induction of IκB-ε, the preferential inhibitor protein of c-Rel dimers. The 6-fold higher c-Rel/IκB-ε ratio found in MCF-7 cells when compared with HeLa cells, may be a contributing factor for the cell-type dependent modulation of c-Rel by H2O2. Our results suggest that H2O2 might have an important cell-type specific role in the regulation of c-Rel-dependent processes, e.g. cancer or wound healing.
Asunto(s)
Peróxido de Hidrógeno/metabolismo , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-rel/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Células HeLa , Humanos , Proteínas I-kappa B/metabolismo , Células MCF-7 , Inhibidor NF-kappaB alfa , Proteínas Proto-Oncogénicas/metabolismo , Transducción de SeñalRESUMEN
Hydrogen peroxide (H2O2) is able to diffuse across biomembranes but, when cells are exposed to external H2O2, the fast consumption of H2O2 inside the cells due to H2O2-removing enzymes provides the driving force for setting up a H2O2 gradient across the plasma membrane. Knowing this gradient is fundamental to standardize studies with H2O2 as for the same extracellular H2O2 concentration cells with different H2O2 gradients may be exposed to different intracellular H2O2 concentrations. Here, we present the kinetic background behind the establishment of the H2O2 gradient and show how the gradient can be determined experimentally using the principle of enzyme latency. Furthermore, we discuss some of the caveats that may arise when determining the H2O2 gradient. Finally, we describe detailed protocols for the experimental determination of the H2O2 gradient across the plasma membrane in Saccharomyces cerevisiae cells and in mammalian cell lines.
Asunto(s)
Glutatión Peroxidasa/química , Peróxido de Hidrógeno/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Algoritmos , Calibración , Pruebas de Enzimas/normas , Peróxido de Hidrógeno/química , Cinética , Oxidación-Reducción , Estándares de Referencia , Saccharomyces cerevisiae/metabolismoRESUMEN
The most common mechanism described for the activation of the transcription factor Nrf2 is based on the inhibition of its degradation in the cytosol followed by its translocation to the nucleus. Recently, Nrf2 de novo synthesis was proposed as an additional mechanism for the rapid upregulation of Nrf2 by hydrogen peroxide (H2O2). Here, we describe a detailed protocol, including solutions, pilot experiments, and experimental setups, which allows exploring the role of H2O2, delivered either as a bolus or as a steady state, in endogenous Nrf2 translocation and synthesis. We also show experimental data, illustrating that H2O2 effects on Nrf2 activation in HeLa cells are strongly dependent both on the H2O2 concentration and on the method of H2O2 delivery. The de novo synthesis of Nrf2 is triggered within 5min of exposure to low concentrations of H2O2, preceding Nrf2 translocation to the nucleus which is slower. Evidence of de novo synthesis of Nrf2 is observed only for low H2O2 steady-state concentrations, a condition that is prevalent in vivo. This study illustrates the applicability of the steady-state delivery of H2O2 to uncover subtle regulatory effects elicited by H2O2 in narrow concentration and time ranges.
Asunto(s)
Núcleo Celular/efectos de los fármacos , Citosol/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Factor 2 Relacionado con NF-E2/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Western Blotting , Núcleo Celular/metabolismo , Citosol/metabolismo , Células HeLa , Humanos , Peróxido de Hidrógeno/análisis , Peróxido de Hidrógeno/metabolismo , Factor 2 Relacionado con NF-E2/agonistas , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/aislamiento & purificación , Transporte de Proteínas/efectos de los fármacos , Transducción de Señal , Activación TranscripcionalRESUMEN
NF-κB is a transcription factor that plays key roles in health and disease. Learning how this transcription factor is regulated by hydrogen peroxide (H2O2) has been slowed down by the lack of methodologies suitable to obtain quantitative data. Literature is abundant with apparently contradictory information on whether H2O2 activates or inhibits NF-κB. There is increasing evidence that H2O2 is not just a generic modulator of transcription factors and signaling molecules but becomes a specific regulator of individual genes. Here, we describe a detailed protocol to obtain rigorous quantitative data on the effect of H2O2 on members of the NF-κB/Rel and IκB families, in which H2O2 is delivered as a steady-state addition instead of the usual bolus addition. Solutions, pilot experiments, and experimental set-ups are fully described. In addition, we outline a protocol to measure the impact of alterations in the promoter κB regions on the H2O2 regulation of the expression of individual genes. As important as evaluating the effects of H2O2 alone is the evaluation of the modulation elicited by this oxidant on cytokine regulation of NF-κB. We illustrate this for the cytokine tumor necrosis factor alpha.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Peróxido de Hidrógeno/metabolismo , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Glucosa/metabolismo , Glucosa Oxidasa/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , FN-kappa B/agonistas , FN-kappa B/genética , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas c-rel/genética , Proteínas Proto-Oncogénicas c-rel/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Hydrogen peroxide (H2O2) is a ubiquitous biological molecule whose wide range of biological functions depends on its concentration. In this chapter, we compare the delivery of H2O2 to cells as (1) a single initial dose (bolus addition); (2) a continuous source using, for example, glucose oxidase; and (3) a steady state, in which H2O2 concentration is kept constant during the assay. Both the bolus addition and the use of a continuous source of H2O2 have as outcome concentration profiles of H2O2 that are dependent on experimental conditions and that are difficult to reproduce from the information that is usually revealed in the experimental section of most research articles. On the other hand, the outcome of delivering H2O2 as a steady state is a concentration profile that is independent of experimental conditions. The implementation of the steady state starts with the determination of the kinetics of H2O2 consumption in the system under study. Then, the amount of glucose oxidase needed to produce H2O2 at a rate that matches the rate of its consumption by cells at the desired H2O2 steady-state concentration is calculated. The setup of the steady state is initiated by adding this amount of glucose oxidase simultaneously with the desired concentration of H2O2. Because H2O2 consumption and delivery rates are matched, the initial H2O2 concentration added is kept constant during the assay. Detailed explanations on how to implement the steady state, including H2O2 measurements and adjustments in the amount of H2O2 or glucose oxidase during the assay, are described.
Asunto(s)
Técnicas Biosensibles/métodos , Peróxido de Hidrógeno/metabolismo , Animales , Catalasa/metabolismo , Electrodos , Glucosa Oxidasa/metabolismo , Glutatión Peroxidasa/metabolismo , Humanos , Oxígeno/metabolismoRESUMEN
In Saccharomyces cerevisiae, adaptation to hydrogen peroxide (H2O2) decreases plasma membrane permeability to H2O2, changes its lipid composition and reorganizes ergosterol-rich microdomains by a still unknown mechanism. Here we show, by a quantitative analysis of the H2O2-induced adaptation effect on the S. cerevisiae plasma membrane-enriched fraction proteome, using two-dimensional gel electrophoresis, that 44 proteins are differentially expressed. Most of these proteins were regulated at a post-transcriptional level. Fourteen of these proteins contain redox-sensitive cysteine residues and nine proteins are associated with lipid and vesicle traffic. In particular, three proteins found in eisosomes and in the eisosome-associated membrane compartment occupied by Can1p were up-regulated (Pil1p, Rfs1p and Pst2p) during adaptation to H2O2. Survival studies after exposure to lethal H2O2 doses using yeast strains bearing a gene deletion corresponding to proteins associated to lipid and vesicle traffic demonstrated for the first time that down-regulation of Kes1p, Vps4p and Ynl010wp and up-regulation of Atp1 and Atp2 increases resistance to H2O2. Moreover, for the pil1Δ strain, H2O2 at low levels produces a hormetic effect by increasing proliferation. In conclusion, these data further confirms the plasma membrane as an active cellular site during adaptation to H2O2 and shows that proteins involved in lipid and vesicle traffic are important mediators of H2O2 adaptation.
Asunto(s)
Peróxido de Hidrógeno/farmacología , Proteoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Adaptación Fisiológica/efectos de los fármacos , Adaptación Fisiológica/fisiología , Membrana Celular/efectos de los fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/genética , Proteoma/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Taking into account published contradictory results concerning the regulation of fatty acid synthase (Fas) by H(2)O(2), we carried out a systematic study where two methods of H(2)O(2) delivery (steady-state and bolus addition) and the effect of a wide range of H(2)O(2) concentrations were investigated. A decrease in Fas activity was observed for cells exposed to 100 and 150µM H(2)O(2) in a steady-state, while a bolus addition of the same H(2)O(2) concentrations did not alter Fas activity. Similar results were observed for the mRNA levels of FAS1, the gene that encodes Fas subunit ß. However, the exposure to a steady-state 50µM H(2)O(2) dose lead to an increase in FAS1 mRNA levels, showing a biphasic modulation of Fas by H(2)O(2). The results obtained emphasize that cellular effects of H(2)O(2) can vary over a narrow range of concentrations. Therefore, a tight control of H(2)O(2) exposure, which can be achieved by exposing H(2)O(2) in a steady-state, is important for cellular studies of H(2)O(2)-dependent redox regulation.
Asunto(s)
Ácido Graso Sintasas/metabolismo , Peróxido de Hidrógeno/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismoRESUMEN
The plasma membrane of Saccharomyces cerevisiae was studied using the probes trans-parinaric acid and diphenylhexatriene. Diphenylhexatriene anisotropy is a good reporter of global membrane order. The fluorescence lifetimes of trans-parinaric acid are particularly sensitive to the presence and nature of ordered domains, but thus far they have not been measured in yeast cells. A long lifetime typical of the gel phase (>30 ns) was found in wild-type (WT) cells from two different genetic backgrounds, at 24 and 30 °C, providing the first direct evidence for the presence of gel domains in living cells. To understand their nature and location, the study of WT cells was extended to spheroplasts, the isolated plasma membrane, and liposomes from total lipid and plasma membrane lipid extracts (with or without ergosterol extraction by cyclodextrin). It is concluded that the plasma membrane is mostly constituted by ordered domains and that the gel domains found in living cells are predominantly at the plasma membrane and are formed by lipids. To understand their composition, strains with mutations in sphingolipid and ergosterol metabolism and in the glycosylphosphatidylinositol anchor remodeling pathway were also studied. The results strongly indicate that the gel domains are not ergosterol-enriched lipid rafts; they are mainly composed of sphingolipids, possibly inositol phosphorylceramide, and contain glycosylphosphatidylinositol-anchored proteins, suggesting an important role in membrane traffic and signaling, and interactions with the cell wall. The abundance of the sphingolipid-enriched gel domains was inversely related to the cellular membrane system global order, suggesting their involvement in the regulation of membrane properties.
Asunto(s)
Microdominios de Membrana/química , Saccharomyces cerevisiae/química , Esferoplastos/química , Esfingolípidos/química , Difenilhexatrieno/química , Ácidos Grasos Insaturados/química , Colorantes Fluorescentes/química , Microdominios de Membrana/metabolismo , Saccharomyces cerevisiae/metabolismo , Esferoplastos/metabolismo , Esfingolípidos/metabolismoRESUMEN
BACKGROUND: The reversible oxidation of protein SH groups has been considered to be the basis of redox regulation by which changes in hydrogen peroxide (H2O2) concentrations may control protein function. Several proteins become S-glutathionylated following exposure to H2O2 in a variety of cellular systems. In yeast, when using a high initial H2O2 dose, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as the major target of S-glutathionylation which leads to reversible inactivation of the enzyme. GAPDH inactivation by H2O2 functions to reroute carbohydrate flux to produce NADPH. Here we report the effect of low regulatory H2O2 doses on GAPDH activity and expression in Saccharomyces cerevisiae. RESULTS: A calibrated and controlled method of H2O2 delivery - the steady-state titration - in which cells are exposed to constant, low, and known H2O2 concentrations, was used in this study. This technique, contrary to the common bolus addition, allows determining which H2O2 concentrations trigger specific biological responses. This work shows that both in exponential- and stationary-phase cells, low regulatory H2O2 concentrations induce a large upregulation of catalase, a fingerprint of the cellular oxidative stress response, but GAPDH oxidation and the ensuing activity decrease are only observed at death-inducing high H2O2 doses. GAPDH activity is constant upon incubation with sub-lethal H2O2 doses, but in stationary-phase cells there is a differential response in the expression of the three GAPDH isoenzymes: Tdh1p is strongly upregulated while Tdh2p/Tdh3p are slightly downregulated. CONCLUSIONS: In yeast GAPDH activity is largely unresponsive to low to moderate H2O2 doses. This points to a scenario where (a) cellular redoxins efficiently cope with levels of GAPDH oxidation induced by a vast range of sub-lethal H2O2 concentrations, (b) inactivation of GAPDH cannot be considered a sensitive biomarker of H2O2-induced oxidation in vivo. Since GAPDH inactivation only occurs at cell death-inducing high H2O2 doses, GAPDH-dependent rerouting of carbohydrate flux is probably important merely in pathophysiological situations. This work highlights the importance of studying H2O2-induced oxidative stress using concentrations closer to the physiological for determining the importance of protein oxidation phenomena in the regulation of cellular metabolism.
Asunto(s)
Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Peróxido de Hidrógeno/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Animales , Ciclo Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/antagonistas & inhibidores , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Oxidación-Reducción , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Hydrogen peroxide (H2O2) has been implicated in the regulation of the transcription factor NF-kappaB, a key regulator of the inflammatory process and adaptive immunity. However, no consensus exists regarding the regulatory role played by H2O2. We discuss how the experimental methodologies used to expose cells to H2O2 produce inconsistent results that are difficult to compare, and how the steady-state titration with H2O2 emerges as an adequate tool to overcome these problems. The redox targets of H2O2 in the NF-kappaB pathway--from the membrane to the post-translational modifications in both NF-kappaB and histones in the nucleus--are described. We also review how H2O2 acts as a specific regulator at the level of the single gene, and briefly discuss the implications of this regulation for human health in the context of kappaB polymorphisms. In conclusion, after near 30 years of research, H2O2 emerges not as an inducer of NF-kappaB, but as an agent able to modulate the activation of the NF-kappaB pathway by other agents. This modulation is generic at the level of the whole pathway but specific at the level of the single gene. Therefore, H2O2 is a fine-tuning regulator of NF-kappaB-dependent processes, as exemplified by its dual regulation of inflammation.
Asunto(s)
Peróxido de Hidrógeno/metabolismo , FN-kappa B/metabolismo , Núcleo Celular/metabolismo , Humanos , Interleucina-1/metabolismo , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Activación Transcripcional , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
In Saccharomyces cerevisiae, the rate of hydrogen peroxide (H(2)O(2)) diffusion through the plasma membrane decreases during adaptation to H(2)O(2) by a still unknown mechanism. Here, adaptation to H(2)O(2) was observed to modulate rapidly the expression of genes coding for enzymes involved in ergosterol and lipid metabolism. Adaptation to H(2)O(2) also alters plasma membrane lipid composition. The main changes were the following: (a) there was a decrease in oleic acid (30%) and in the ratio between unsaturated and saturated long-chain fatty acids; (b) the phosphatidylcholine:phosphatidylethanolamine ratio increased threefold; (c) sterol levels were unaltered but there was an increased heterogeneity of sterol-rich microdomains and increased ordered domains; (d) the levels of the sterol precursor squalene increased twofold, in agreement with ERG1 gene down-regulation; and (e) C26:0 became the major very long chain fatty acid owing to an 80% decrease in 2-hydroxy-C26:0 levels and a 50% decrease in C20:0 levels, probably related to the down-regulation of fatty acid elongation (FAS1, FEN1, SUR4) and ceramide synthase (LIP1, LAC1) genes. Therefore, H(2)O(2) leads to a reorganization of the plasma membrane microdomains, which may explain the lower permeability to H(2)O(2), and emerges as an important regulator of lipid metabolism and plasma membrane lipid composition.
Asunto(s)
Membrana Celular/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Microdominios de Membrana/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Animales , Membrana Celular/microbiología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Canal de Potasio ERG1 , Ergosterol/metabolismo , Canales de Potasio Éter-A-Go-Go/genética , Canales de Potasio Éter-A-Go-Go/metabolismo , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Regulación de la Expresión Génica , Microdominios de Membrana/microbiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ácido Oléico/análisis , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Fosfatidilcolinas/análisis , Fosfatidiletanolaminas/análisis , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Escualeno/análisis , Esteroles/análisisRESUMEN
We recently observed that H2O2 regulates inflammation via upexpression of a few NF-kappaB-dependent genes, while leaving expression of most NF-kappaB-dependent genes unaltered. Here we test the hypothesis that this differential gene expression depends on the apparent affinity of kappaB sites in the gene-promoter regions toward NF-kappaB. Accordingly, cells were transfected with three reporter plasmids containing kappaB sequences with different affinities for NF-kappaB. It was observed that the lower the affinity, the higher the range of TNF-alpha concentrations where H2O2 upregulated gene expression. Mathematical models reproduced the key experimental observations indicating that H2O2 upregulation ceased when NF-kappaB fully occupied the kappaB sites. In vivo, it is predicted that genes with high-affinity sites remain insensitive to H2O2, whereas genes with lower-affinity sites are upregulated by H2O2. In conclusion, a simple chemical mechanism is at the root of a complex biologic process such as differential gene expression caused by H2O2.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , FN-kappa B/fisiología , Secuencia de Bases , Cartilla de ADN , Genes Reporteros , Células HeLa , Humanos , Transcripción GenéticaRESUMEN
In Saccharomyces cerevisiae, the diffusion rate of hydrogen peroxide (H2O2) through the plasma membrane decreases during adaptation to H2O2 by means of a mechanism that is still unknown. Here, evidence is presented that during adaptation to H2O2 the anisotropy of the plasma membrane increases. Adaptation to H2O2 was studied at several times (15min up to 90min) by applying the steady-state H2O2 delivery model. For wild-type cells, the steady-state fluorescence anisotropy increased after 30min, or 60min, when using 2-(9-anthroyloxy) stearic acid (2-AS), or diphenylhexatriene (DPH) membrane probe, respectively. Moreover, a 40% decrease in plasma membrane permeability to H2O2 was observed at 15min with a concomitant two-fold increase in catalase activity. Disruption of the ergosterol pathway, by knocking out either ERG3 or ERG6, prevents the changes in anisotropy during H2O2 adaptation. H2O2 diffusion through the plasma membrane in S. cerevisiae cells is not mediated by aquaporins since the H2O2 permeability constant is not altered in the presence of the aquaporin inhibitor mercuric chloride. Altogether, these results indicate that the regulation of the plasma membrane permeability towards H2O2 is mediated by modulation of the biophysical properties of the plasma membrane.
Asunto(s)
Permeabilidad de la Membrana Celular/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/efectos de los fármacos , Adaptación Fisiológica/efectos de los fármacos , Anisotropía , Acuaporinas/metabolismo , Transporte Biológico/efectos de los fármacos , Fenómenos Biofísicos , Biofisica , Fluidez de la Membrana/efectos de los fármacos , Mutación/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Changes in plasma membrane permeability caused by H2O2 were recently found to be involved in the adaptation to H2O2, but the mechanism responsible for this change remains largely unknown. Here this mechanism was addressed and two lines of evidence showed for the first time that fatty acid synthase (Fas) plays a key role during the cellular response of Saccharomyces cerevisiae to H2O2: (1) adaptation was associated with a decrease in both Fas expression and activity; (2) more importantly, decreasing Fas activity by 50% through deletion of one of the FAS alleles increased the resistance to lethal doses of H2O2. The mechanism by which a decrease of Fas expression causes a higher resistance to H2O2 was not fully elucidated. However, the fas1Delta strain plasma membrane had large increases in the levels of lignoceric acid (C24:0) (40%) and cerotic acid (C26:0) (50%), suggesting that alterations in the plasma membrane composition are involved. Very-long-chain fatty acids (VLCFA) through interdigitation or by modulating formation of lipid rafts may decrease the overall or localized plasma membrane permeability to H2O2, respectively, thus conferring a higher resistance to H2O2.
Asunto(s)
Ácido Graso Sintasas/metabolismo , Peróxido de Hidrógeno/farmacología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Membrana Celular/química , Regulación hacia Abajo , Farmacorresistencia Fúngica/genética , Ácido Graso Sintasas/genética , Ácidos Grasos/análisis , Ácidos Grasos/metabolismo , Eliminación de Gen , Expresión Génica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Although the germicide role of H(2)O(2) released during inflammation is well established, a hypothetical regulatory function, either promoting or inhibiting inflammation, is still controversial. In particular, after 15 years of highly contradictory results it remains uncertain whether H(2)O(2) by itself activates NF-kappaB or if it stimulates or inhibits the activation of NF-kappaB by proinflammatory mediators. We investigated the role of H(2)O(2) in NF-kappaB activation using, for the first time, a calibrated and controlled method of H(2)O(2) delivery--the steady-state titration--in which cells are exposed to constant, low, and known concentrations of H(2)O(2). This technique contrasts with previously applied techniques, which disrupt cellular redox homeostasis and/or introduce uncertainties in the actual H(2)O(2) concentration to which cells are exposed. In both MCF-7 and HeLa cells, H(2)O(2) at extracellular concentrations up to 25 microM did not induce significantly per se NF-kappaB translocation to the nucleus, but it stimulated the translocation induced by TNF-alpha. For higher H(2)O(2) doses this stimulatory role shifts to an inhibition, which may explain published contradictory results. The stimulatory role was confirmed by the observation that 12.5 microM H(2)O(2), a concentration found during inflammation, increased the expression of several proinflammatory NF-kappaB-dependent genes induced by TNF-alpha (e.g., IL-8, MCP-1, TLR2, and TNF-alpha). The same low H(2)O(2) concentration also induced the anti-inflammatory gene coding for heme oxygenase-1 (HO-1) and IL-6. We propose that H(2)O(2) has a fine-tuning regulatory role, comprising both a proinflammatory control loop that increases pathogen removal and an anti-inflammatory control loop, which avoids an exacerbated harmful inflammatory response.
Asunto(s)
Núcleo Celular/metabolismo , Peróxido de Hidrógeno/farmacología , FN-kappa B/metabolismo , Oxidantes/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/inmunología , Núcleo Celular/inmunología , Quimiocina CCL2/biosíntesis , Quimiocina CCL2/inmunología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Células HeLa , Hemo-Oxigenasa 1 , Homeostasis/efectos de los fármacos , Homeostasis/inmunología , Humanos , Peróxido de Hidrógeno/agonistas , Peróxido de Hidrógeno/inmunología , Inflamación/inmunología , Inflamación/metabolismo , Interleucina-6/biosíntesis , Interleucina-6/inmunología , Interleucina-8/biosíntesis , Interleucina-8/inmunología , FN-kappa B/inmunología , Oxidantes/agonistas , Oxidación-Reducción/efectos de los fármacos , Receptor Toll-Like 2/biosíntesis , Receptor Toll-Like 2/inmunología , Factor de Necrosis Tumoral alfa/agonistas , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Contrary to what is widely believed, recent published results show that H2O2 does not freely diffuse across biomembranes. The fast removal of H2O2 by antioxidant enzymes is able to generate a gradient if H2O2 is produced in a different compartment from that containing the enzymes (Antunes, F., and Cadenas, E. (2000) FEBS Lett. 475, 121-126). In this work, we extended these studies and tested whether an active regulation of biomembranes permeability characteristics is part of the cell response to oxidative stress. Using Saccharomyces cerevisiae as a model, we showed that: (a) H2O2 gradients across the plasma membrane are formed upon exposure to external H2O2; (b) there is a correlation between the magnitude of the gradients and the resistance to H2O2; (c) there is not a correlation between the intracellular capacity to remove H2O2 and the resistance to H2O2; (d) the plasma membrane permeability to H2O2 decreases by a factor of two upon acquisition of resistance to this agent by pre-exposing cells either to nonlethal doses of H2O2 or to cycloheximide, an inhibitor of protein synthesis; and (e) erg3Delta and erg6Delta mutants, which have impaired ergosterol biosynthesis pathways, show higher plasma membrane permeability to H2O2 and are more sensitive to H2O2. Altogether, the regulation of the plasma membrane permeability to H2O2 emerged as a new mechanism by which cells respond and adapt to H2O2. The consequences of the results to cellular redox compartmentalization and to the origin and evolution of the eukaryotic cell are discussed.
Asunto(s)
Membrana Celular/metabolismo , Peróxido de Hidrógeno/metabolismo , Saccharomyces cerevisiae/metabolismo , Antioxidantes/farmacología , Catalasa/metabolismo , Supervivencia Celular , Cicloheximida/farmacología , Citocromo-c Peroxidasa/metabolismo , Ergosterol/química , Cinética , Oxidación-Reducción , Estrés Oxidativo , Permeabilidad , Inhibidores de la Síntesis de la Proteína/farmacología , Esferoplastos/metabolismo , Temperatura , Factores de TiempoRESUMEN
Gene expression of three antioxidant enzymes, Mn superoxide dismutase (MnSOD), Cu,Zn superoxide dismutase (Cu,ZnSOD), and glutathione reductase (GR) was investigated in stationary phase Saccharomyces cerevisiae during menadione-induced oxidative stress. Both GR and Cu,ZnSOD mRNA steady state levels increased, reaching a plateau at about 90 min exposure to menadione. GR mRNA induction was higher than that of Cu,ZnSOD (about 14-fold and 9-fold after 90 min, respectively). A different pattern of response was obtained for MnSOD mRNA, with a peak at about 15 min (about 8-fold higher) followed by a decrease to a plateau approximately 4-fold higher than the control value. However, these increased mRNA levels did not result in increased protein levels and activities of these enzymes. Furthermore, exposure to menadione decreased MnSOD activity to half its value, indicating that the enzyme is partially inactivated due to oxidative damage. Cu,ZnSOD protein levels were increased 2-fold, but MnSOD protein levels were unchanged after exposure to menadione in the presence of the proteolysis inhibitor phenylmethylsulfonyl fluoride. These results indicate that the rates of Cu,ZnSOD synthesis and proteolysis are increased, while the rates of MnSOD synthesis and proteolysis are unchanged by exposure to menadione. Also, the translational efficiency for both enzymes is probably decreased, since increases in protein levels when proteolysis is inhibited do not reflect the increases in mRNA levels. Our results indicate that oxidative stress modifies MnSOD, Cu,ZnSOD, and GR gene expression in a complex way, not only at the transcription level but also at the post-transcriptional, translational, and post-translational levels.