Regulation of Ras proteins by reactive nitrogen species

Ras GTPases have been a subject of intense investigation since the early 1980s, when single point mutations in Ras were shown to cause deregulated cell growth control. Subsequently, Ras was identified as the most prevalent oncogene found in human cancer. Ras proteins regulate a host of pathways involved in cell growth, differentiation, and apoptosis by cycling between inactive GDP-bound and active GTP-bound states. Regulation of Ras activity is controlled by cellular factors that alter guanine nucleotide cycling. Oncogenic mutations prevent protein regulatory factors from down-regulating Ras activity, thereby maintaining Ras in a chronically activated state. The central dogma in the field is that protein modulatory factors are the primary regulators of Ras activity. Since the mid-1990s, however, evidence has accumulated that small molecule reactive nitrogen species (RNS) can also influence Ras guanine nucleotide cycling. Herein, we review the basic chemistry behind RNS formation and discuss the mechanism through which various RNS enhance nucleotide exchange in Ras proteins. In addition, we present studies that demonstrate the physiological relevance of RNS-mediated Ras activation within the context of immune system function, brain function, and cancer development. We also highlight future directions and experimental methods that may enhance our ability to detect RNS-mediated activation in cell cultures and in vivo. The development of such methods may ultimately pave new directions for detecting and elucidating how Ras proteins are regulated by redox species, as well as for targeting redox-activated Ras in cancer and other disease states.     

Down regulation of superoxide dismutases and glutathione peroxidase by reactive oxygen and nitrogen species

The levels of antioxidative enzymes are regulated by gene expressions as well as by post-translational modifications. Although their functions are to scavenge reactive oxygen (ROS) and nitrogen species (RNS), they may also be targets of various oxidants. When ROS and RNS modify the functions of antioxidative enzymes, especially glutathione peroxidase, they may induce apoptotic cell death in susceptible cells. It is conceivable, therefore, that at least a part of the apoptotic pathways mediated by ROS and RNS may be associated with modification of the redox regulation of cellular functions due to elevations of such substances. In this article we review recent findings about the effects of various oxidative conditions associated with alteration of these antioxidative enzymes and the concomitant cellular damage induced.     

Down regulation of superoxide dismutases and glutathione peroxidase by reactive oxygen and nitrogen species

The levels of antioxidative enzymes are regulated by gene expressions as well as by post-translational modifications. Although their functions are to scavenge reactive oxygen (ROS) and nitrogen species (RNS), they may also be targets of various oxidants. When ROS and RNS modify the functions of antioxidative enzymes, especially glutathione peroxidase, they may induce apoptotic cell death in susceptible cells. It is conceivable, therefore, that at least a part of the apoptotic pathways mediated by ROS and RNS may be associated with modification of the redox regulation of cellular functions due to elevations of such substances. In this article we review recent findings about the effects of various oxidative conditions associated with alteration of these antioxidative enzymes and the concomitant cellular damage induced.     

Fyn and JAK2 mediate Ras activation by reactive oxygen species.

Reactive oxygen species (ROS) activate Ras and the extracellular signal-regulated kinase (ERK) cascade. Because JAK2 is a critical mediator for Ras/Raf/ERK activation by several hormones, we examined the role of JAK2 in ROS signal events. H(2)O(2) stimulated JAK2 activity in fibroblasts with peak at 2-5 min. To determine the specific role of Src and Fyn as mediators of JAK2 activation and its downstream events, we used fibroblasts derived from transgenic mice deficient in Src (Src-/-) or Fyn (Fyn-/-). H(2)O(2)-stimulated JAK2 activity was completely inhibited in Fyn-/- cells. Shc tyrosine phosphorylation and Ras activation by H(2)O(2) were also significantly reduced in Fyn-/- cells, but not altered in Src-/- cells. Activation of JAK2 was restored when Fyn-/- cells were transfected with B-Fyn but not with Src. Inhibiting JAK2 activity with the specific inhibitor AG-490 prevented H(2)O(2) stimulated Shc and Ras activation. H(2)O(2)-mediated ERK1/2 activation in Fyn-/- cells and AG-490 treated cells was completely inhibited at an early time (5 min), but not at late times (20-40 min) after stimulation. These results define a new redox-sensitive pathway for Ras activation and rapid ERK1/2 activation, which is mediated by Fyn and JAK2.     

Cell signaling by reactive nitrogen and oxygen species in atherosclerosis

The production of reactive oxygen and nitrogen species has been implicated in atherosclerosis principally as means of damaging low-density lipoprotein that in turn initiates the accumulation of cholesterol in macrophages. The diversity of novel oxidative modifications to lipids and proteins recently identified in atherosclerotic lesions has revealed surprising complexity in the mechanisms of oxidative damage and their potential role in atherosclerosis. Oxidative or nitrosative stress does not completely consume intracellular antioxidants leading to cell death as previously thought. Rather, oxidative and nitrosative stress have a more subtle impact on the atherogenic process by modulating intracellular signaling pathways in vascular tissues to affect inflammatory cell adhesion, migration, proliferation, and differentiation. Furthermore, cellular responses can affect the production of nitric oxide, which in turn can strongly influence the nature of oxidative modifications occurring in atherosclerosis. The dynamic interactions between endogenous low concentrations of oxidants or reactive nitrogen species with intracellular signaling pathways may have a general role in processes affecting wound healing to apoptosis, which can provide novel insights into the pathogenesis of atherosclerosis.     

Clinical aspects of reactive oxygen and nitrogen species

Endothelial dysfunction in the setting of cardiovascular risk factors, such as hypercholesterolaemia, hypertension, diabetes mellitus and chronic smoking, as well as in the setting of heart failure, has been shown to be at least partly dependent on the production of reactive oxygen species in endothelial and/or smooth muscle cells and the adventitia, and the subsequent decrease in vascular bioavailability of NO. Superoxide-producing enzymes involved in increased oxidative stress within vascular tissue include NAD(P)H-oxidase, xanthine oxidase and endothelial nitric oxide synthase in an uncoupled state. Recent studies indicate that endothelial dysfunction of peripheral and coronary resistance and conductance vessels represents a strong and independent risk factor for future cardiovascular events. Ways to reduce endothelial dysfunction include risk-factor modification and treatment with substances that have been shown to reduce oxidative stress and, simultaneously, to stimulate endothelial NO production, such as inhibitors of angiotensin-converting enzyme or the statins. In contrast, in conditions where increased production of reactive oxygen species, such as superoxide, in vascular tissue is established, treatment with NO, e.g. via administration of nitroglycerin, results in a rapid development of endothelial dysfunction, which may worsen the prognosis in patients with established coronary artery disease.     

Oxidative depolymerization of polysaccharides by reactive oxygen/nitrogen species

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are constantly produced and are tightly regulated to maintain a redox balance (or homeostasis) together with antioxidants (e.g. superoxide dismutase and glutathione) under normal physiological circumstances. These ROS/RNS have been shown to be critical for various biological events including signal transduction, aging, apoptosis, and development. Despite the known beneficial effects, an overproduction of ROS/RNS in the cases of receptor-mediated stimulation and disease-induced oxidative stress can inflict severe tissue damage. In particular, these ROS/RNS are capable of degrading macromolecules including proteins, lipids and nucleic acids as well as polysaccharides, and presumably lead to their dysfunction. The purpose of this review is to highlight (1) chemical mechanisms related to cell-free and cell-based depolymerization of polysaccharides initiated by individual oxidative species; (2) the effect of ROS/RNS-mediated depolymerization on the successive cleavage of the glycosidic linkage of polysaccharides by glycoside hydrolases; and (3) the potential biological outcome of ROS/RNS-mediated depolymerization of polysaccharides.     

Mass spectrometry of protein modifications by reactive oxygen and nitrogen species

The modification of proteins by reactive oxygen and nitrogen species plays an important role in various biologic processes involving protein activation and inactivation, protein translocation and turnover during signal transduction, stress response, proliferation, and apoptosis. Recent advances in protein and peptide separation and mass spectrometry provide increasingly sophisticated tools for the quantitative analysis of such protein modifications, which are absolutely necessary for their correlation with biologic phenomena. The present review focuses specifically on the qualitative and quantitative mass spectrometric analysis of the most common protein modifications caused by reactive oxygen and nitrogen species in vivo and in vitro and details a case study on a membrane protein the sarco/endoplasmic reticulum Ca-ATPase (SERCA).     

Ras proteins induce senescence by altering the intracellular levels of reactive oxygen species

Human diploid fibroblasts eventually lose the capacity to replicate in culture and enter a viable but nonproliferative state of senescence. Recently, it has been demonstrated that retroviral-mediated gene transfer into primary fibroblasts of an activated ras gene (V12ras) rapidly accelerates development of the senescent phenotype. Using this in vitro system, we have sought to define the mediators of Ras-induced senescence. We demonstrate that expression of V12Ras results in an increase in intracellular and in particular, mitochondrial reactive oxygen species. The ability of V12Ras to induce growth arrest and senescence is shown to be partially inhibited by coexpression of an activated rac1 gene. A more dramatic rescue of V12Ras-expressing cells is demonstrated when the cells are placed in a low oxygen environment, a condition in which reactive oxygen species production is inhibited. In addition, in a 1% oxygen environment, Ras is unable to trigger an increase in the level of the cyclin-dependent kinase inhibitor p21 or to activate the senescent program. Under normoxic (20% O2) conditions, the V12Ras senescent phenotype is demonstrated to be unaffected by scavengers of superoxide but rescued by scavengers of hydrogen peroxide. These results suggest that in normal diploid cells, Ras proteins regulate oxidant production and that a rise in intracellular H2O2 represents a critical signal mediating replicative senescence.     

Roles of reactive oxygen and nitrogen species in pain

Peroxynitrite (PN; ONOO⁻) and its reactive oxygen precursor superoxide (SO; O₂^•−) are critically important in the development of pain of several etiologies including pain associated with chronic use of opiates such as morphine (also known as opiate-induced hyperalgesia and antinociceptive tolerance). This is now an emerging field in which considerable progress has been made in terms of understanding the relative contributions of SO, PN, and nitroxidative stress in pain signaling at the molecular and biochemical levels. Aggressive research in this area is poised to provide the pharmacological basis for development of novel nonnarcotic analgesics that are based upon the unique ability to selectively eliminate SO and/or PN. As we have a better understanding of the roles of SO and PN in pathophysiological settings, targeting PN may be a better therapeutic strategy than targeting SO. This is because, unlike PN, which has no currently known beneficial role, SO may play a significant role in learning and memory [1]. Thus, the best approach may be to spare SO while directly targeting its downstream product, PN. Over the past 15 years, our team has spearheaded research concerning the roles of SO and PN in pain and these results are currently leading to the development of solid therapeutic strategies in this important area.     

Interaction of reactive oxygen and nitrogen species with proteins

Free radicals and reactive oxygen or nitrogen species generated during oxidative stress and as by-products of normal cellular metabolism may damage all types of biological molecules. Proteins are major initial targets in cell. Reactions of a variety of free radicals and reactive oxygen and nitrogen species with proteins can lead to oxidative modifications of proteins such as protein hydroperoxides formation, hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, oxidation of sulfhydryl groups, oxidation of methionine residues, conversion of some amino acid residues into carbonyl groups, cleavage of the polypeptide chain and formation of cross-linking bonds. Such modifications of proteins leading to loss of their function (enzymatic activity), accumulation and inhibition of their degradation have been observed in several human diseases, aging, cell differentiation and apoptosis. Formation of specific protein oxidation products may be used as biomarkers of oxidative stress.     

The production of reactive oxygen and nitrogen species by skeletal muscle.

Skeletal muscle has been recognized as a potential source for generation of reactive oxygen and nitrogen species for more than 20 years. Initial investigations concentrated on the potential role of mitochondria as a major source for generation of superoxide as a "by-product" of normal oxidative metabolism, but recent studies have identified multiple subcellular sites, where superoxide or nitric oxide are generated in regulated and controlled systems in response to cellular stimuli. Full evaluation of the factors regulating these processes and the functions of the reactive oxygen species generated are important in understanding the redox biology of skeletal muscle.     

Mitochondria: regulators of signal transduction by reactive oxygen and nitrogen species

The functional role of mitochondria in cell physiology has previously centered around metabolism, with oxidative phosphorylation playing a pivotal role. Recently, however, this perspective has changed significantly with the realization that mitochondria are active participants in signal transduction pathways, not simply the passive recipients of injunctions from the rest of the cell. In this review the emerging role of the mitochondrion in cell signaling is discussed in the context of cytochrome c release, hydrogen peroxide formation from the respiratory chain, and the nitric oxide-cytochrome c oxidase signaling pathway.     

Apoptosis and macrophage clearance of neutrophils: regulation by reactive oxygen species

Inflammation is a beneficial host response to foreign challenge involving numerous soluble factors and cell types, including polymorphonuclear granulocytes or neutrophils. Programmed cell death (apoptosis) of neutrophils has been documented in vitro as well as in vivo, and is thought to be important for the resolution of inflammation, as this process allows for engulfment and removal of senescent cells prior to their necrotic disintegration. Studies in recent years have begun to unravel the mechanism of macrophage clearance of apoptotic cells, and evidence has accrued for a critical role of externalization and oxidation of plasma membrane phosphatidylserine, and its subsequent recognition by macrophage receptors, in this process. Activated neutrophils generate vast amounts of reactive oxygen species for the purpose of killing ingested micro-organisms, and these reactive metabolites may also modulate the life-span, as well as the clearance, of the neutrophil itself. This review aims to address the latter topic, as well as to summarize current knowledge on the molecular mechanisms of neutrophil apoptosis and macrophage clearance of these cells at the site of inflammation.     

Antimicrobial reactive oxygen and nitrogen species: concepts and controversies

Phagocyte-derived reactive oxygen and nitrogen species are of crucial importance for host resistance to microbial pathogens. Decades of research have provided a detailed understanding of the regulation, generation and actions of these molecular mediators, as well as their roles in resisting infection. However, differences of opinion remain with regard to their host specificity, cell biology, sources and interactions with one another or with myeloperoxidase and granule proteases. More than a century after Metchnikoff first described phagocytosis, and more than four decades after the discovery of the burst of oxygen consumption that is associated with microbial killing, the seemingly elementary question of how phagocytes inhibit, kill and degrade microorganisms remains controversial. This review updates the reader on these concepts and the topical questions in the field.     

Metal chelators react also with reactive oxygen and nitrogen species

Popular chelators (desferrioxamine, SIH, EDTA, EGTA, DTPA, and NTA) were demonstrated to have antioxidant properties, being able to reduce ABTS radical cation and react with peroxyl radicals, peroxynitrite, and hypochlorite. Desferrioxamine and SIH were most potent antioxidants in all cases. These results point to the necessity of a careful interpretation of experiments in which the inhibition of free radical reactions by antioxidants is used as a proof of involvement of metal ions in a reaction.     

Biochemical reactivity of melatonin with reactive oxygen and nitrogen species

Melatonin (N-acetyl-5-methoxytryptamine), an endogenously produced indole found throughout the animal kingdom, was recently reported, using a variety of techniques, to be a scavenger of a number of reactive oxygen and reactive nitrogen species both in vitro and in vivo. Initially, melatonin was discovered to directly scavenge the high toxic hydroxyl radical (*OH). The methods used to prove the interaction of melatonin with the *OH included the generation of the radical using Fenton reagents or the ultraviolet photolysis of hydrogen peroxide (H202) with the use of spin-trapping agents, followed by electron spin resonance (ESR) spectroscopy, pulse radiolysis followed by ESR, and several spectrofluorometric and chemical (salicylate trapping in vivo) methodologies. One product of the reaction of melatonin with the *OH was identified as cyclic 3-hydroxymelatonin (3-OHM) using high-performance liquid chromatography with electrochemical (HPLC-EC) detection, electron ionization mass spectrometry (EIMS), proton nuclear magnetic resonance (1H NMR) and COSY 1H NMR. Cyclic 3-OHM appears in the urine of humans and other mammals and in rat urine its concentration increases when melatonin is given exogenously or after an imposed oxidative stress (exposure to ionizing radiation). Urinary cyclic 3-OHM levels are believed to be a biomarker (footprint molecule) of in vivo *OH production and its scavenging by melatonin. Although the data are less complete, besides the *OH, melatonin in cell-free systems has been shown to directly scavenge H2O2, singlet oxygen (1O2) and nitric oxide (NO*), with little or no ability to scavenge the superoxide anion radical (O2*-) In vitro, melatonin also directly detoxifies the peroxynitrite anion (ONOO-) and/or peroxynitrous acid (ONOOH), or the activated form of this molecule, ONOOH*; the product of the latter interaction is proposed to be 6-OHM. How these in vitro findings relate to the in vivo antioxidant actions of melatonin remains to be established. The ability of melatonin to scavenge the lipid peroxyl radical (LOO*) is debated. The weight of the evidence is that melatonin is probably not a classic chain-breaking antioxidant, since its ability to scavenge the LOO* seems weak. Its ability to reduce lipid peroxidation may stem from its function as a preventive antioxidant (scavenging initiating radicals), or yet unidentified actions. In sum, in vitro melatonin acts as a direct free radical scavenger with the ability to detoxify both reactive oxygen and reactive nitrogen species; in vivo, it is an effective pharmacological agent in reducing oxidative damage under conditions in which excessive free radical generation is believed to be involved.