Carla Real

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.

The extracellular matrix modulates H2O2 degradation and redox signaling in endothelial cells

The molecular processes that are crucial for cell function, such as proliferation, migration and survival, are regulated by hydrogen peroxide (H2O2). Although environmental cues, such as growth factors, regulate redox signaling, it was still unknown whether the ECM, a component of the cell microenvironment, had a function in this process. Here, we showed that the extracellular matrix (ECM) differently regulated H2O2 consumption by endothelial cells and that this effect was not general for all types of cells. The analysis of biophysical properties of the endothelial cell membrane suggested that this modification in H2O2 consumption rates was not due to altered membrane permeability. Instead, we found that the ECM regulated GPx activity, a known H2O2 scavenger. Finally, we showed that the extent of PTEN oxidation was dependent on the ECM, indicating that the ECM was able to modulate H2O2-dependent protein oxidation. Thus, our results unraveled a new mechanism by which the ECM regulates endothelial cell function by altering redox balance. These results pinpoint the ECM as an important component of redox-signaling.

Hydrogen peroxide regulates cell adhesion through the redox sensor RPSA

To become metastatic, a tumor cell must acquire new adhesion properties that allow migration into the surrounding connective tissue, transmigration across endothelial cells to reach the blood stream and, at the site of metastasis, adhesion to endothelial cells and transmigration to colonize a new tissue. Hydrogen peroxide (H2O2) is a redox signaling molecule produced in tumor cell microenvironment with high relevance for tumor development. However, the molecular mechanisms regulated by H2O2 in tumor cells are still poorly known. The identification of H2O2-target proteins in tumor cells and the understanding of their role in tumor cell adhesion are essential for the development of novel redox-based therapies for cancer. In this paper, we identified Ribosomal Protein SA (RPSA) as a target of H2O2 and showed that RPSA in the oxidized state accumulates in clusters that contain specific adhesion molecules. Furthermore, we showed that RPSA oxidation improves cell adhesion efficiency to laminin in vitro and promotes cell extravasation in vivo. Our results unravel a new mechanism for H2O2-dependent modulation of cell adhesion properties and identify RPSA as the H2O2 sensor in this process. This work indicates that high levels of RPSA expression might confer a selective advantage to tumor cells in an oxidative environment.

Opi1p translocation to the nucleus is regulated by hydrogen peroxide in Saccharomyces cerevisiae

During exposure of yeast cells to low levels of hydrogen peroxide (H2O2), 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 H2O2. 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 H2O2 in concentrations inducing adaptation regulates Opi1p function. We found that, in the presence of H2O2, 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 H2O2‐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 H2O2 in yeast cells. Copyright

Therapeutic activity of superoxide dismutase-containing enzymosomes on rat liver ischaemia-reperfusion injury followed by magnetic resonance microscopy

&NA; Liver ischaemia‐reperfusion injury (IRI) may occur during hepatic surgery and is unavoidable in liver transplantation. Superoxide dismutase enzymosomes (SOD‐enzymosomes), liposomes where SOD is at the liposomal surface expressing enzymatic activity in intact form without the need of liposomal disruption, were developed with the aim of having a better insight into its antioxidant therapeutic outcome in IRI. We also aimed at validating magnetic resonance microscopy (MRM) at 7 T as a tool to follow IRI. SOD‐enzymosomes were characterized and tested in a rat ischaemia‐reperfusion model and the therapeutic outcome was compared with conventional long circulating SOD liposomes and free SOD using biochemical liver injury biomarkers, histology and MRM. MRM results correlated with those obtained using classical biochemical biomarkers of liver injury and liver histology. Moreover, MRM images suggested that the therapeutic efficacy of both SOD liposomal formulations used was related to prevention of peripheral biliary ductular damage and disrupted vascular architecture. Therefore, MRM at 7 T is a useful technique to follow IRI. SOD‐enzymosomes were more effective than conventional liposomes in reducing liver ischaemia‐reperfusion injury and this may be due to a short therapeutic window. Graphical abstract Figure. No caption available.

Noncoding RNAs as Critical Players in Regulatory Accuracy, Redox Signaling, and Immune Cell Functions

The transcriptome of multicellular organisms is much more complex than initially thought because it includes a large number of noncoding RNAs (ncRNAs). Data regarding ncRNAs suggest that organism complexity better correlates with the percentage of each genome that is transcribed into these molecules. The most studied classes of ncRNAs are short interfering RNAs, microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), and long noncoding RNAs (lncRNAs). In this chapter, we review the biogenesis pathways and general functions of miRNAs, piRNAs, and lncRNAs. We focus on the roles of miRNAs and lncRNAs in gene expression regulation, centering on redox signaling and immune cell development, and highlight some implications for human pathologies. Finally, we analyze current knowledge concerning the use of ncRNAs in diagnosis, prognosis, and therapeutics, and discuss their role in the development of the immune system and the regulatory functions of H 2 O 2 during the course of metazoan evolution.

Hydrogen peroxide regulates angiogenesis-related factors in tumor cells

Tumor angiogenesis is required for tumor development and growth, and is regulated by several factors including ROS. H2O2 is a ROS with an important role in cell signaling, but how H2O2 regulates tumor angiogenesis is still poorly understood. We have xenografted tumor cells with altered levels of H2O2 by catalase overexpression into zebrafish embryos to study redox-induced tumor neovascularization. We found that vascular recruitment and invasion were impaired if catalase was overexpressed. In addition, the overexpression of catalase altered the transcriptional levels of several angiogenesis-related factors in tumor cells, including TIMP-3 and THBS1. These two anti-angiogenic factors were found to be H2O2-regulated by two different mechanisms: TIMP-3 expression in a cell-autonomous manner; and, THBS1 expression that was non-cell-autonomous. Our work shows that intracellular H2O2 regulates the expression of angiogenic factors and the formation of a vessel network. Understanding the molecular mechanisms that govern this multifunctional effect of H2O2 on tumor angiogenesis could be important for the development of more efficient anti-angiogenic therapies.

Data on intracellular localization of RPSA upon alteration of its redox state

Ribosomal Protein SA (RPSA), a component of the 40S ribosomal subunit, was identified as a H2O2 target in HeLa cells [1]. In order to analyze the intracellular localization of RPSA in different redox states we overexpressed wild-type RPSA (RPSAwt) or RPSA containing two cysteine to serine residue substitutions at positions 148 and 163 (RPSAmut) in HeLa cells. The transfected cells were exposed to H2O2 or N-acetylcysteine (NAC) and RPSA subcellular localization was assessed by immunofluorescence in permeabilized cells. In addition, co-immunofluorescence for RPSA and Ribosomal Protein S6 (RPS6) was performed in cells overexpressing RPSAwt or RPSAmut. Finally, the ribosomal expression of endogenous RPSA in the presence or absence of H2O2 was analyzed by Western blot. The data presented in this work is related to the research article entitled “Hydrogen peroxide regulates cell adhesion through the redox sensor RPSA” [1].