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.

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.

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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 transparinaric 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.