Hydrogen peroxide and peroxiredoxins in redox regulation of intracellular signaling

A growing body of evidence indicates that hydrogen peroxide is generated in response to ligand-receptor interaction and is involved in redox-regulation of signaling pathways in various cell types. The mechanism of redox-regulation is based on post-translational modification of key regulatory proteins that contain essential cysteinyl residues at their catalytic sites. Hydrogen peroxide acts as a chemical mediator that affects signaling through reversible oxidation of thiol groups of essential cysteins, which results in changes in protein catalytic activity. The accumulation and propagation of H₂O₂ signal in a cell is regulated by antioxidant proteins peroxiredoxins. The catalytic cycle of peroxiredoxins enables them to keep a low resting level of H₂O₂ in a cell, while the hyperoxidation-reduction cycle allows for H₂O₂ bursts during signal transduction. H₂O₂, being a small, diffusible, highly reactive molecule generated and destroyed in enzymatic reactions and capable of regulating the phosphorylation-dephosphorylation events in a cell, meets all major criteria of the second messenger in signal transduction but the criterion of specificity. How a toxic molecule that is potentially dangerous to all biomolecules in a cell can specifically relay information through signaling cascades is discussed in this review.     

Hydrogen peroxide mediates oxidant-dependent stimulation of arterial smooth muscle L-type calcium channels

Changes in calcium and redox homeostasis influence multiple cellular processes. Dysregulation of these signaling modalities is associated with pathology in cardiovascular, neuronal, endocrine, and other physiological systems. Calcium and oxidant signaling mechanisms are frequently inferred to be functionally related. To address and clarify this clinically relevant issue in the vasculature we tested the hypothesis that the ubiquitous reactive oxygen molecule hydrogen peroxide mediates oxidant-dependent stimulation of cerebral arterial smooth muscle L-type calcium channels. Using a combinatorial approach including intact arterial manipulations, electrophysiology, and total internal reflection fluorescence imaging, we found that application of physiological levels of hydrogen peroxide to isolated arterial smooth muscle cells increased localized calcium influx through L-type calcium channels. Similarly, oxidant-dependent stimulation of L-type calcium channels by the vasoconstrictor ANG II was abolished by intracellular application of catalase. Catalase also prevented ANG II from increasing localized subplasmalemmal sites of increased oxidation previously associated with colocalized calcium influx through L-type channels. Furthermore, catalase largely attenuated the contractile response of intact cerebral arterial segments to ANG II. In contrast, enhanced dismutation of superoxide to hydrogen peroxide with SOD had no effect on ANG II-dependent stimulation of L-type calcium channels. From these data we conclude that hydrogen peroxide is important for oxidant-dependent regulation of smooth muscle L-type calcium channels and arterial function. These data also support the emerging concept of hydrogen peroxide as a biologically relevant oxidant second messenger in multiple cell types with a diverse array of physiological functions.     

Redox signaling: thiol chemistry defines which reactive oxygen and nitrogen species can act as second messengers

Except for the role of NO in the activation of guanylate cyclase, which is well established, the involvement of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in signal transduction remains controversial, despite a large body of evidence suggestive of their participation in a variety of signaling pathways. Several problems have limited their acceptance as signaling molecules, with the major one being the difficulty in identifying the specific targets for each pathway and the chemical reactions supporting reversible oxidation of these signaling components, consistent with a second messenger role for ROS and RNS. Nevertheless, it has become clear that cysteine residues in the thiolate (i.e., ionized) form that are found in some proteins can be specific targets for reaction with H₂O₂ and RNS. This review focuses on the chemistry of the reversible oxidation of those thiolates, with a particular emphasis on the critical thiolate found in protein tyrosine phosphatases as an example. hydrogen peroxide; thiolate; nitrosothiol; nitric oxide; signal transduction     

Distribution of oxidized and HNE-modified proteins in U87 cells

Protein modification is one of the important processes during oxidative stress. This modification of proteins is either due to direct oxidation of proteins by various oxidants or due to secondary modification by lipid peroxidation products, e.g. 4-hydroxynonenal. In the here presented work we compare the intracellular distribution of protein modification products after treatment of human U87 astrocytoma cells with hydrogen peroxide or HNE. The treatment with hydrogen peroxide leads mainly to a cytosolic formation of oxidized proteins whereas HNE treatment is forming HNE-adducts throughout the cell. Therefore, we concluded that HNE diffusion distance in cells enables this lipid peroxidation product to act as a second messenger within the cell and on the other hand is the reason for the genotoxic properties of this compound.     

Are free radicals involved in thiol-based redox signaling?

Cells respond to many stimuli by transmitting signals through redox-regulated pathways. It is generally accepted that in many instances signal transduction is via reversible oxidation of thiol proteins, although there is uncertainty about the specific redox transformations involved. The prevailing view is that thiol oxidation occurs by a two electron mechanism, most commonly involving hydrogen peroxide. Free radicals, on the other hand, are considered as damaging species and not generally regarded as important in cell signaling. This paper examines whether it is justified to dismiss radicals or whether they could have a signaling role. Although there is no direct evidence that radicals are involved in transmitting thiol-based redox signals, evidence is presented that they are generated in cells when these signaling pathways are activated. Radicals produce the same thiol oxidation products as two electron oxidants, although by a different mechanism, and at this point radical-mediated pathways should not be dismissed. There are unresolved issues about how radical mechanisms could achieve sufficient selectivity, but this could be possible through colocalization of radical-generating and signal-transducing proteins. Colocalization is also likely to be important for nonradical signaling mechanisms and identification of such associations should be a priority for advancing the field.     

The essential requirement for superoxide radical and nitric oxide formation for normal physiological function and healthy aging

Contrary to the dogma that superoxide anion and hydrogen peroxide formation are highly deleterious to cell function and healthy aging, we suggest this premise is flawed. Superoxide anion and hydrogen peroxide formation are essential to normal cellular function; they constitute a second messenger system absolutely required for the regulation of the metabolome. Embraced within this regulation is the modulation of cellular redox poise, bioenergy output, gene expression and cell differentiation. A key component in the overall process is coenzyme Q10 whose prooxidant function through the formation of superoxide anion and hydrogen peroxide is a major factor in the overall processes. The free radical gas, nitric oxide (similarly to superoxide anion), functions in the regulation of a wide range of cell systems. As part of the normal physiological process, superoxide anion and NO function separately and interactively as second messengers. Superoxide anion and nitric oxide play an intrinsic role in the regulated ordered turnover of proteins, rather than randomly cause protein damage and their inactivation. The proposition that metabolic free radical formation is unequivocally deleterious to cell function is rebutted; their toxicity as primary effectors in the aging process has been overemphasized. The concept that a dietary supplement of high concentrations of small-molecule antioxidants is a prophylactic/amelioration therapy for the aging process and age-associated diseases is questioned as to its clinical validity.     

In vivo levels of mitochondrial hydrogen peroxide increase with age in mtDNA mutator mice

In mtDNA mutator mice, mtDNA mutations accumulate leading to a rapidly aging phenotype. However, there is little evidence of oxidative damage to tissues, and when analyzed ex vivo, no change in production of the reactive oxygen species (ROS) superoxide and hydrogen peroxide by mitochondria has been reported, undermining the mitochondrial oxidative damage theory of aging. Paradoxically, interventions that decrease mitochondrial ROS levels in vivo delay onset of aging. To reconcile these findings, we used the mitochondria‐targeted mass spectrometry probe MitoB to measure hydrogen peroxide within mitochondria of living mice. Mitochondrial hydrogen peroxide was the same in young mutator and control mice, but as the mutator mice aged, hydrogen peroxide increased. This suggests that the prolonged presence of mtDNA mutations in vivo increases hydrogen peroxide that contributes to an accelerated aging phenotype, perhaps through the activation of pro‐apoptotic and pro‐inflammatory redox signaling pathways.     

Multiple G-protein-coupled pathways inhibit N-type Ca channels of neurons

Muscarinic receptors depress Ca2+ currents in superior cervical ganglion neurons by two signaling pathways. One is sensitive to pertussis toxin and acts rapidly by a membrane-delimited pathway on the channels. The other is not sensitive to pertussis toxin and acts more slowly through an unknown second messenger. These pathways are shared with several other agonists.     

Hydrogen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding, systemin, and methyl jasmonate

The systemic accumulation of both hydrogen peroxide (H(2)O(2)) and proteinase inhibitor proteins in tomato leaves in response to wounding was inhibited by the NADPH oxidase inhibitors diphenylene iodonium (DPI), imidazole, and pyridine. The expression of several defense genes in response to wounding, systemin, oligosaccharides, and methyl jasmonate also was inhibited by DPI. These genes, including those of four proteinase inhibitors and polyphenol oxidase, are expressed within 4 to 12 hr after wounding. However, DPI did not inhibit the wound-inducible expression of genes encoding prosystemin, lipoxygenase, and allene oxide synthase, which are associated with the octadecanoid signaling pathway and are expressed 0.5 to 2 hr after wounding. Accordingly, treatment of plants with the H(2)O(2)-generating enzyme glucose oxidase plus glucose resulted in the induction of only the later-expressed defensive genes and not the early-expressed signaling-related genes. H(2)O(2) was cytochemically detected in the cell walls of vascular parenchyma cells and spongy mesophyll cells within 4 hr after wounding of wild-type tomato leaves, but not earlier. The cumulative results suggest that active oxygen species are generated near cell walls of vascular bundle cells by oligogalacturonide fragments produced by wound-inducible polygalacturonase and that the resulting H(2)O(2) acts as a second messenger for the activation of defense genes in mesophyll cells. These data provide a rationale for the sequential, coordinated, and functional roles of systemin, jasmonic acid, oligogalacturonides, and H(2)O(2) signals for systemic signaling in tomato plants in response to wounding.     

Methylene blue directly oxidizes glutathione without the intermediate formation of hydrogen peroxide

Methylene blue stimulates the oxidation of glutathione in red blood cells in vitro and in vivo. This oxidation has been attributed to hydrogen peroxide that is generated from the autooxidation of leucomethylene blue arising from the reduction of methylene blue by NADPH. In this report we present evidence that methylene blue directly oxidizes glutathione and that oxidation of glutathione by hydrogen peroxide is a secondary reaction. Moreover, superoxide dismutase has no effect on the oxidation. Under aerobic conditions, methylene blue oxidizes glutathione 30 times faster than the spontaneous autooxidation of glutathione. Under anaerobic conditions the stoichiometry of the reaction of methylene blue with glutathione supports a direct chemical reaction. The reaction rates between glutathione and methylene blue suggest a second order reaction over the conditions tested. That neither oxygen radical formation nor significant amounts of hydrogen peroxide are produced by methylene blue, even in the presence of added glucose, is further confirmed by the failure to detect significant amounts of lipid peroxidation products, or hemolysis, in red blood cells incubated with the dye.     

Involvement of sphingomyelinases in TNF signaling pathways

Sphingomyelin (N-acylsphingosin-1-phosphorylcholine) is a phospholipid preferentially found in the plasma membrane of mammalian cells. Signaling through the sphingomyelin pathway is associated with generation of ceramide, which acts as a second messenger in activating a variety of cellular functions. Ceramide belongs to the group of sphingosine-based lipid second messenger molecules that are critically involved in the regulation of signal transduction of diverse cell surface membrane receptors. The emerging picture suggests that coupling of ceramide to specific signaling cascades is both stimulus- and cell type-specific and depends on the subcellular topology of its production. Following membrane receptor triggering, neutral and acid isoforms of sphingomyelinases are rapidly activated generating ceramide through sphingomyelin hydrolysis. Here the molecular mechanisms of TNF-induced activation of sphingomyelinases and the functional consequences of ceramide generation will be discussed.     

Proteomic profile of reversible protein oxidation using PROP, purification of reversibly oxidized proteins

Signal transduction pathways that are modulated by thiol oxidation events are beginning to be uncovered, but these discoveries are limited by the availability of relatively few analytical methods to examine protein oxidation compared to other signaling events such as protein phosphorylation. We report here the coupling of PROP, a method to purify reversibly oxidized proteins, with the proteomic identification of the purified mixture using mass spectrometry. A gene ontology (GO), KEGG enrichment and Wikipathways analysis of the identified proteins indicated a significant enrichment in proteins associated with both translation and mRNA splicing. This methodology also enabled the identification of some of the specific cysteine residue targets within identified proteins that are reversibly oxidized by hydrogen peroxide treatment of intact cells. From these identifications, we determined a potential consensus sequence motif associated with oxidized cysteine residues. Furthermore, because we identified proteins and specific sites of oxidation from both abundant proteins and from far less abundant signaling proteins (e.g. hepatoma derived growth factor, prostaglandin E synthase 3), the results suggest that the PROP procedure was efficient. Thus, this PROP-proteomics methodology offers a sensitive means to identify biologically relevant redox signaling events that occur within intact cells.     

An investigation into copper catalyzed D-penicillamine oxidation and subsequent hydrogen peroxide generation

D-Penicillamine is a potent copper (Cu) chelating agent. D-Pen reduces Cu(II) to Cu(I) in the process of chelation while at the same time being oxidized to D-penicillamine disulfide. It has been proposed that hydrogen peroxide is generated during this process. However, definitive experimental proof that hydrogen peroxide is generated remains lacking. Thus, the major aims of these studies were to confirm and quantitatively assess the in vitro production of hydrogen peroxide during copper catalyzed D-penicillamine oxidation. The potential cytotoxic effect of hydrogen peroxide generation was also investigated in vitro against MCF-7 human breast cancer cells. Cell cytotoxicity resulting from the incubation of D-penicillamine with copper was compared to that of D-penicillamine, copper and hydrogen peroxide. The mechanism of copper catalyzed D-penicillamine oxidation and simultaneous hydrogen peroxide production was investigated as a function of time, concentration of cupric sulfate or ferric chloride, temperature, pH, anaerobic condition and chelators such as ethylenediaminetetraacetic acid and bathocuproinedisulfonic acid. A simple, sensitive and rapid HPLC assay was developed to simultaneously detect D-penicillamine, its major oxidation product D-penicillamine disulfide, and hydrogen peroxide in a single run. Hydrogen peroxide was shown to be generated in a concentration dependent manner as a result of D-penicillamine oxidation in the presence of cupric sulfate. Chelators such as ethylenediaminetetraacetic acid and bathocuproinedisulfonic acid were able to inhibit D-penicillamine oxidation. The incubation of MCF-7 human breast cancer cells with D-penicillamine plus cupric sulfate resulted in the production of reactive oxygen species within the cell and cytotoxicity that was comparable to free hydrogen peroxide.     

Regulatory control of human cytosolic branched-chain aminotransferase by oxidation and S-glutathionylation and its interactions with redox sensitive neuronal proteins

Redox regulation of proteins through oxidation and S-thiolation are important regulatory processes, acting in both a protective and adaptive role in the cell. In the current study, we investigated the sensitivity of the neuronal human cytosolic branched-chain aminotransferase (hBCATc) protein to oxidation and S-thiolation, with particular attention focused on functionality and modulation of its CXXC motif. Thiol specific reagents showed significant redox cycling between the reactive thiols and the TNB anion, and using NEM, four of the six reactive thiols are critical to the functionality of hBCATc. Site-directed mutagenesis studies supported these findings where a reduced kcat (ranging from 50-70% of hBCATc) for C335S, C338S, C335/8S, and C221S, respectively, followed by a modest effect on C242S was observed. However, only the thiols of the CXXC motif (C335 and C338) were directly involved in the reversible redox regulation of hBCATc through oxidation (with a loss of 40-45% BCAT activity on air oxidation alone). Concurrent with these findings, under air oxidation, the X-ray crystallography structure of hBCATc showed a disulphide bond between C335 and C338. Further oxidation of the other four thiols was not evident until levels of hydrogen peroxide were elevated. S-thiolation experiments of hBCATc exposed to GSH provided evidence for significant recycling between GSH and the thiols of hBCATc, which implied that under reducing conditions GSH was operating as a thiol donor with minimal S-glutathionylation. Western blot analysis of WT hBCATc and mutant proteins showed that as the ratio of GSH:GSSG decreased significant S-glutathionylation occurred (with a further loss of 20% BCAT activity), preferentially at the thiols of the CXXC motif, suggesting a shift in function toward a more protective role for GSH. Furthermore, the extent of S-glutathionylation increased in response to oxidative stress induced by hydrogen peroxide potentially through a C335 sulfenic acid intermediate. Deglutathionylation of hBCATc-SSG using the GSH/glutaredoxin system provides evidence that this protein may play an important role in cellular redox regulation. Moreover, redox associations between hBCATc and several neuronal proteins were identified using targeted proteomics. Thus, our data provides strong evidence that the reactive thiol groups, in particular the thiols of the CXXC motif, play an integral role in redox regulation and that hBCATc has redox mediated associations with several neuronal proteins involved in G-protein cell signaling, indicating a novel role for hBCATc in cellular redox control.     

Mitochondrial network determines intracellular ROS dynamics and sensitivity to oxidative stress through switching inter-mitochondrial messengers

Oxidative stresses caused by reactive oxygen species (ROS) can induce rapid depolarization of inner mitochondrial membrane potential and subsequent impairment of oxidative phosphorylation. Damaged mitochondria produce more ROS, especially the superoxide anion (O(2)(-)) and hydrogen peroxide (H(2)O(2)), which potentiate mitochondria-driven ROS propagation, so-called ROS-induced ROS release (RIRR), via activation of an inter-mitochondria signaling network. Therefore, loss of function in only a fraction of mitochondria might eventually affect cell viability through this positive feedback loop. Since ROS are very short-lived molecules in the biological milieu, mitochondrial network dynamics, such as density, number, and spatial distribution, can affect mitochondria-driven ROS propagation. To address this issue, we developed a mathematical model using an agent-based modeling approach, and tested the effect of mitochondrial network dynamics on RIRR for mitochondria under various conditions. Simulation results show that the intracellular ROS signaling pattern, such as ROS propagation speed and oxidative stress vulnerability, are critically affected by mitochondrial network dynamics. Mitochondrial network dynamics of mitochondrial distribution, density, activity, and size can mediate inter-mitochondrial signaling under certain conditions and determine the identity of the ROS signaling pattern. We further elucidated the potential mechanism of these actions, i.e., conversion of major messenger molecules involved in ROS signaling. If the average distance between neighboring mitochondria is large or mitochondrial distribution becomes randomized, messenger molecule of the ROS signaling network can be switched from O(2)(-) to H(2)O(2). In this case, mitochondria-driven ROS propagation is efficiently blocked by introduction of excess cytosolic glutathione peroxidase 1, while introduction of cytosolic superoxide dismutase has no effect. Together, these results suggest that mitochondrial network dynamics is a major determinant for cellular responses to RIRR through changing the key messenger molecules.     

On the functional significance of substrate accessibility in the photosynthetic water oxidation mechanism

Recent evidence suggests that after selective perturbation of the protein structure in photosystem II, hydrogen peroxide is formed at the water oxidation catalytic site instead of molecular oxygen. In this communication, we review the interpretation of these observations and elaborate on the hypothesis that an essential factor in determining the end-product of photosynthetic water oxidation is one of substrate accessibility. It is argued that normally the access of water to the catalytic site is controlled by a hydrophobic domain in the surrounding protein matrix and that the production of O₂ is optimized by an ordered binding of the two substrate water molecules. It is proposed that upon perturbation of the hydrophobic domain (for example, through the removal of various extrinsic proteins) the catalytic site becomes exposed to excess water from the external solvent phase. As a consequence, additional water binds at the catalytic site during intermediate oxidation steps and undergoes a partial oxidation reaction to form hydrogen peroxide. The importance of water accessibility to the structure/function relationships of photosystem II is discussed, particularly with respect to photoinhibitory damage through the formation of hydrogen peroxide.     

Hydrogen peroxide linked to lysine oxidase activity facilitates biofilm differentiation and dispersal in several gram-negative bacteria

The marine bacterium Pseudoalteromonas tunicata produces an antibacterial and autolytic protein, AlpP, which causes death of a subpopulation of cells during biofilm formation and mediates differentiation, dispersal, and phenotypic variation among dispersal cells. The AlpP homologue (LodA) in the marine bacterium Marinomonas mediterranea was recently identified as a lysine oxidase which mediates cell death through the production of hydrogen peroxide. Here we show that AlpP in P. tunicata also acts as a lysine oxidase and that the hydrogen peroxide generated is responsible for cell death within microcolonies during biofilm development in both M. mediterranea and P. tunicata. LodA-mediated biofilm cell death is shown to be linked to the generation of phenotypic variation in growth and biofilm formation among M. mediterranea biofilm dispersal cells. Moreover, AlpP homologues also occur in several other gram-negative bacteria from diverse environments. Our results show that subpopulations of cells in microcolonies also die during biofilm formation in two of these organisms, Chromobacterium violaceum and Caulobacter crescentus. In all organisms, hydrogen peroxide was implicated in biofilm cell death, because it could be detected at the same time as the killing occurred, and the addition of catalase significantly reduced biofilm killing. In C. violaceum the AlpP-homologue was clearly linked to biofilm cell death events since an isogenic mutant (CVMUR1) does not undergo biofilm cell death. We propose that biofilm killing through hydrogen peroxide can be linked to AlpP homologue activity and plays an important role in dispersal and colonization across a range of gram-negative bacteria.