H. Susana Marinho

Hydrogen peroxide sensing, signaling and regulation of transcription factors

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 × 103 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.

Regulation of antioxidant enzymes gene expression in the yeast Saccharomyces cerevisiae during stationary phase

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.

H2O2 induces rapid biophysical and permeability changes in the plasma membrane of Saccharomyces cerevisiae.

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.

Gel Domains in the Plasma Membrane of Saccharomyces cerevisiae HIGHLY ORDERED, ERGOSTEROL-FREE, AND SPHINGOLIPID-ENRICHED LIPID RAFTS

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.

Modulation of plasma membrane lipid profile and microdomains by H2O2 in Saccharomyces cerevisiae.

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.

ROLE OF GLUTATHIONE PEROXIDASE AND PHOSPHOLIPID HYDROPEROXIDE GLUTATHIONE PEROXIDASE IN THE REDUCTION OF LYSOPHOSPHOLIPID HYDROPEROXIDES

1-linoleoyl lysophosphatidylcholine hydroperoxide is a substrate of GSH peroxidase (GPx) both purified from bovine erythrocytes and nonpurified from rat liver. The initial reaction rate for bovine erythrocyte GPx with 1-linoleoyl lysophosphatidylcholine hydroperoxide is about 76 and 95% of the reaction rate for hydrogen peroxide and linoleic acid hydroperoxide respectively. For rat liver GPx these initial reaction rates are about 66 and 75%, respectively. The rate constants for the reaction of GPx with 1-linoleoyl lysophosphatidylcholine hydroperoxide were calculated to be approximately 3 x 10(7) M-1s-1 and approximately 2 x 10(6) M-1s-1 for the bovine erythrocyte and the rat liver enzymes, respectively. By using kinetic models of lipid peroxidation we found by simulation that: (1) the main source of lysophospholipid hydroperoxides in vivo is the peroxidation of lysophospholipids, both in mitochondrial inner membranes and in endoplasmic reticulum; (2) a specialized enzyme able to reduce directly lysophospholipid hydroperoxides is important for the reduction of these hydroperoxides, because the detoxification of these species mediated by the action of acyl ester bond cleaving enzymes is not efficient; (3) the reduction through GPx predominates over phospholipid hydroperoxide glutathione peroxidase (PHGPx) in mitochondrial inner membranes and in the cytosolic phase of the endoplasmic reticulum; (4) in the luminal phase of endoplasmic reticulum PHGPx is predominant.

H2O2 delivery to cells: steady-state versus bolus addition.

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.

Activation of Nrf2 by H2O2: De Novo Synthesis Versus Nuclear Translocation

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.

Glutathione metabolism in hepatomous liver of rats treated with diethylnitrosamine

Glutathione metabolism was studied in rat liver during diethylnitrosamine (DEN) carcinogenesis. Some studies were also made in foetal rat liver. Endogenous GSH and non-protein thiols concentrations are increased in DEN-treated rats when compared to non-treated rats but no differences were found in cysteine, total thiols and protein thiols concentration. In foetal liver GSH concentration is only 35% of that in DEN-treated rat liver. The activities of several enzymes involved in glutathione metabolism are changed in DEN-treated rats. gamma-Glutamyl transferase activity and cysteine formation from GSH by liver homogenates is increased sevenfold. gamma-Glutamylcysteine synthetase activity, initial rate of [35S]cysteine incorporation in gamma-glutamylcysteine and initial rate of GSH formation from [35S]cysteine are increased two-fold. Cytosolic GSH S-transferase activity is increased twofold in DEN-treated rats and so GSH S-conjugates concentration is probably also increased. In foetal rat liver gamma-glutamyl transferase activity is about the same but gamma-glutamylcysteine synthetase activity is only 10% of that in DEN-treated rat liver. The increased GSH concentration in DEN-treated rat liver is probably due to the simultaneous increase in the activities of gamma-glutamyl transferase and gamma-glutamylcysteine synthetase. Blood plasma total glutathione is increased 1.4 times in DEN-treated rats, but no differences are found in GSH hepatic arteriovenous gradient. This associated with the increased gamma-glutamyl transferase activity suggests that sinusoidal GSH efflux is increased in DEN-treated rats.

The cellular steady-state of H2O2: latency concepts and gradients.

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.