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Protein tyrosine nitration - Functional alteration or just a biomarker?
Abstract
Protein 3-nitrotyrosine is a posttranslational modification found in many pathological conditions from acute to chronic diseases. Could 3-nitrotyrosine formation participate on the basis of these diseases or is it just a marker connected with the associated nitroxidative stress? In vitro and in vivo data, including proteomic research, show that protein tyrosine nitration is a selective process where only a small amount of proteins is found nitrated and one or a few tyrosine residues are modified in each. Accumulating data suggest a strong link between protein 3-nitrotyrosine and the mechanism involved in disease development. In this review, we analyze the factors determining protein 3-nitrotyrosine formation, the functional and biological outcome associated with protein tyrosine nitration, and the fate of the nitrated proteins.
... Multiple reports indicate that GS inactivation can occur via oxidants other than ONOOand via alternative mechanisms (46)(47)(48)(49). Furthermore, it is unlikely that only single residues (such as Tyr336) would be selectively nitrated when pure GS is exposed to ONOO -, given the presence of 15 Tyr residues per monomer (43,50,51). In addition, there is limited semi-quantitative or quantitative data with respect to the extent of Tyr336 modification (and also for other residues), which is critical to addressing whether a biologically relevant loss of activity occurs via nitration at this site, especially as Tyr nitration is typically a low yield reaction (37, 50,52). ...
... Furthermore, it is unlikely that only single residues (such as Tyr336) would be selectively nitrated when pure GS is exposed to ONOO -, given the presence of 15 Tyr residues per monomer (43,50,51). In addition, there is limited semi-quantitative or quantitative data with respect to the extent of Tyr336 modification (and also for other residues), which is critical to addressing whether a biologically relevant loss of activity occurs via nitration at this site, especially as Tyr nitration is typically a low yield reaction (37, 50,52). Finally, the possibility that GS oxidation by ONOO -(and other oxidants) may affect protein function by mechanisms other than impaired catalysis (e.g. via protein unfolding and/or aggregation) has not been explored. ...
... Complete HsGS inactivation was obtained with ONOOconcentrations as high as 5 mM ( Fig. 2A), accompanied by an increase in NO2Tyr concentrations from ~3 µM (at 0.1 mM ONOO -) to ≈ 22 µM (at 5 mM ONOO -); the latter corresponds to ~5 NO2Tyr residues per HsGS monomer ( Fig. 2A). To complement the MS/MS analysis presented above, HsGS samples exposed to ONOO - (50,250, 500 and 1000 µM) were digested with trypsin and analyzed for NO2Tyr-containing peptides, through the detection of NO2Tyr-immonium ions (54). This approach identified several of the nitration sites reported above, as well as a previously non-detected nitration site at Tyr171 (Table SI). ...
Glutamine synthetase (GS), which catalyzes the ATP-dependent synthesis of L-glutamine from L-glutamate and ammonia, is a ubiquitous and conserved enzyme that plays a pivotal role in nitrogen metabolism across all life domains. In vertebrates, GS is highly expressed in astrocytes, where its activity sustains the glutamate-glutamine cycle at glutamatergic synapses and is thus essential for maintaining brain homeostasis. In fact, decreased GS levels or activity have been associated with neurodegenerative diseases, with these alterations attributed to oxidative post-translational modifications of the protein, in particular tyrosine nitration. In this study, we expressed and purified human GS (HsGS) and performed an in-depth analysis of its oxidative inactivation by peroxynitrite (ONOO-) in vitro. We found that ONOO- exposure led to a dose-dependent loss of HsGS activity, the oxidation of cysteine, methionine and tyrosine residues and also the nitration of tryptophan and tyrosine residues. Peptide mapping by LC-MS/MS through combined H216O/H218O trypsin digestion identified up to 10 tyrosine nitration sites and five types of dityrosine cross-links; these modifications were further scrutinized by structural analysis. Tyrosine residues 171, 185, 269, 283 and 336 were the main nitration targets; however, tyrosine-to-phenylalanine HsGS mutants revealed that their sole nitration was not responsible for enzyme inactivation. In addition, we observed that ONOO- induced HsGS aggregation and activity loss. Thiol oxidation was a key modification to elicit aggregation, as it was also induced by hydrogen peroxide treatment. Taken together, our results indicate that multiple oxidative events at various sites are responsible for the inactivation and aggregation of human GS.
... In mitochondria, the electron transport chain (CIII/IV) triggers NO production. In of H2O2 and NO2 - (Souza et al., 2008). In general, the bioavailability and bioactivity of ONOOdepend on ...
... there is still no known consensus sequence that guarantees this selectivity (Bartesaghi and Radi, 2018). To 306 date, some common features that apparently affect Y-NO2 formation are known, such as the presence of 307 acidic residues next to the nitration site, the cysteine or methionine residues adjacent to the target tyrosine 308 residue and the presence of loop-forming amino acids like proline or glycine (Souza et al., 2008). Other factors that also influence the nitration process are the centrifugal-centripetal position of the tyrosine residue in the three-dimensional (3D) structure of the protein, the cellular environment and the redox conditions of ...
Reactive nitrogen species are a set of highly reactive molecules that derive from nitric oxide and oxidative metabolism, such as nitrogen dioxide, peroxynitrite, nitroxyl anion, nitrate, among others. The role of reactive nitrogen species in plants has become an area of much scientific interest in recent years. In particular, nitric oxide and peroxynitrite are the main reactive nitrogen species best known for their important interactions with different cellular metabolic processes related to seed germination, plant growth and development, root development, stomatal movements, interaction with phytohormones, reproduction, pollen tube development and fruit ripening. It is also relevant to highlight the important role of nitric oxide as a signalling molecule involved in alleviating toxic effects caused by various biotic and abiotic stresses via intense cellular metabolic reprogramming. Thus, nitric oxide is able to modulate oxidative, antioxidant and phytohormonal metabolisms, among others, as well as ion homeostasis, metal transport and transcriptional factors. With such a background, this chapter summarises recent advances in the metabolic pathways in generating reactive nitrogen species, their bioactivity to react with different cellular molecules, like proteins, nucleic acids and fatty acids, and finally, their role in plant metabolism under physiological and stress conditions.KeywordsAbiotic stressBiotic stressDevelopmentMetabolismNitric oxideNitro-fatty acidsNucleic acid nitrationPeroxynitriteProtein nitrationReactive nitrogen speciesS-nitrosylation
... In the first step, the tyrosyl radical (Tyr • ) is formed through the oxidation of the aromatic ring of Tyr, due to the influence of oxidants (hydroxyl (OH • ) and carbonate (CO 3 •− ) radicals) formed as a result of the decomposition of ONOO − . Next, Tyr • interacts with • NO 2 (formed through the decomposition of ONOO − in the presence of CO 2 ), which results in the formation of 3-nitrotyrosine [128,130,131]. Due to the fact that this mechanism affects the sites of free radical formation, nitrated proteins are usually located close to the compartments of their formation due to the short biological half-life of nitrogen radicals, e.g., ONOO − [132][133][134]. ...
... In most cases, nitration is an irreversible reaction that can usually cause a loss or inhibition of protein function [137,140,141]. Increasing the hydrophobicity of the Tyr residue after nitration also promotes conformational changes [131]. Nitration may affect cell signaling through regulating the level of Tyr phosphorylation. ...
Simple Summary: Nitric oxide (NO) is a key molecule that has an important role in the plant life cycle. It mediates a range of physiological processes and responses to stresses (e.g., drought, salinity, or parasite invasion). Despite many studies, knowledge about NO involvement in these processes is incomplete. This review describes the influence of NO on physiological and biochemical processes and gene expression. It thoroughly discusses the interaction network of NO and other molecules in plant cells. Moreover, it highlights mechanisms of NO-dependent defense response against infestation with Ecdysozoa species, like nematodes, insects, and arachnids. Abstract: Nitric oxide (NO) is an important signaling molecule that is involved in a wide range of physiological processes in plants, including responses to biotic and abiotic stresses. Changes in endogenous NO concentration lead to activation/deactivation of NO signaling and NO-related processes. This paper presents the current state of knowledge on NO biosynthesis and scavenging pathways in plant cells and highlights the role of NO in post-translational modifications of proteins (S-nitrosylation, nitration, and phosphorylation) in plants under optimal and stressful environmental conditions. Particular attention was paid to the interactions of NO with other signaling molecules: reactive oxygen species, abscisic acid, auxins (e.g., indole-3-acetic acid), salicylic acid, and jasmonic acid. In addition, potential common patterns of NO-dependent defense responses against attack and feeding by parasitic and molting Ecdysozoa species such as nematodes, insects, and arachnids were characterized. Our review definitely highlights the need for further research on the involvement of NO in interactions between host plants and Ecdysozoa parasites, especially arachnids.
... [3][4][5] Although low levels of protein tyrosine nitration are detected under basal conditions, there is a significant increase in nitroxidative stress-associated diseases, such as neurodegenerative, inflammatory and cardiovascular conditions. [6,7] In these circumstances, nitration of protein tyrosine residues can induce different effects on protein function. [1,3,4,6,7] For example, nitration of glutathione reductase residues Tyr106 and Tyr114 promotes a loss of function by reducing its activity due to the decreased binding affinity for its substrate. ...
... [6,7] In these circumstances, nitration of protein tyrosine residues can induce different effects on protein function. [1,3,4,6,7] For example, nitration of glutathione reductase residues Tyr106 and Tyr114 promotes a loss of function by reducing its activity due to the decreased binding affinity for its substrate. [8,9] The opposite effect is observed in cytochrome c, [10] protein kinase Cɛ, [11] or αsynuclein, [12] among others, which after nitration would show a new or increased function associated with a significant biological effect. ...
Tyrosine nitration, a post‐translational modification (PTM) that takes place under nitrosative stress conditions, occurs through a non‐enzymatic peroxynitrite‐mediated reaction. Although protein nitration has long been considered an irreversible PTM involved in nitrosative stress‐associated diseases, it has also been suggested to be a regulatory mechanism of signal transduction. Therefore, the development of tools that help to understand this protein modification is of great interest. Herein, we explore a TbIII‐chelating metallopeptide to monitor tyrosine nitration. The luminescence of this probe decreases significantly between its non‐nitrated and nitrated states, and this reduction in the luminescence intensity is directly related to the degree of tyrosine nitration after treatment with peroxynitrite. Remarkably, the luminescence intensity changes after nitration are not affected in the presence of complex biological media, which makes it a promising tool for understanding this protein modification.
... Reduced NO bioavailability promotes an oxidative imbalance by simultaneously enhancing ROS and RNS production and down-regulating key antioxidant enzymes [47]. Increased levels of ROS indicate excessive production of superoxide, which reacts with NO to form peroxynitrite. Peroxynitrite can cause nitration of tyrosine residues on proteins to form nitrotyrosine [47] and has been identified as an indicator or marker of cell damage and inflammation [48][49][50]. The elevated level of nitrotyrosine reflects the degree of tissue damage [49], while the presence of nitrotyrosine in the kidney detected with immunohistochemistry could be associated with progressive deterioration of the kidney [51]. ...
... Increased levels of ROS indicate excessive production of superoxide, which reacts with NO to form peroxynitrite. Peroxynitrite can cause nitration of tyrosine residues on proteins to form nitrotyrosine [47] and has been identified as an indicator or marker of cell damage and inflammation [48][49][50]. The elevated level of nitrotyrosine reflects the degree of tissue damage [49], while the presence of nitrotyrosine in the kidney detected with immunohistochemistry could be associated with progressive deterioration of the kidney [51]. ...
Objective
Background
The main cause of death among patients with malignant hypertension is a kidney failure. The promising field in essential and malignant hypertension therapy could be centered on the amelioration of oxidative stress using antioxidant molecules like resveratrol. Resveratrol is a potent antioxidative agent naturally occurred in many plants that possess health-promoting properties.
Methods
In the present study, we investigated the therapeutic potential of resveratrol, a polyphenol with anti-oxidative activity, in NG-L-Arginine Methyl Ester (L-NAME) treated spontaneously hypertensive rats (SHR) - malignantly hypertensive rats (MHR).
Results
Resveratrol significantly improves oxidative damages by modulation of antioxidant enzymes and suppression of prooxidant factors in the kidney tissue of MHR. Enhanced antioxidant defense in the kidney improves renal function and ameliorates the morphological changes in this target organ. Besides, protective properties of resveratrol are followed by the restoration of the nitrogen oxide (NO) pathway. 4) Conclusion: Antioxidant therapy with resveratrol could represent promising therapeutical approach in hypertension, especially malignant, against kidney damage.
... Nitration of tyrosine residues can profoundly alter not only the protein structure but also the functions, suggesting that protein nitration could be predictive of oxidative cell injury. Indeed, several studies have demonstrated the presence of 3-NT in pathological conditions, including CVD (Ahsan, 2013;Souza et al., 2008). ...
... In the context of HF, data demonstrate the presence of an increased 3-NT plasma level in patients with moderate/severe HF in association with markers of systemic inflammation (Eleuteri et al., 2009), and in particular ceruloplasmin nitration has been evidenced and associated bad prognosis in patients with HF (Cabassi et al., 2014). Under inflammatory conditions, such as in CVD, only 1-10 residues of tyrosine every 100,000 have been shown to be nitrated in plasma proteins, while these levels increase approximately 10 times in tissues, such as atherosclerotic plaques (Souza et al., 2008). ...
The Human Plasma Proteome has always been the most investigated compartment in proteomics‐based biomarker discovery, and is considered the largest and deepest version of the human proteome, reflecting the state of the body in health and disease. Even if efforts have been always dedicated to the refinement of proteomic approaches to investigate more deeply the plasma proteome, it should not be forgotten that also highly abundant plasma proteins, like human serum albumin (HSA), often neglected in these studies, might provide fundamental physiological functions in plasma, and should be better considered. This review summarizes the important roles of HSA in the context of cardiovascular diseases (CVD), and in particular in heart failure. Notwithstanding much attention has been historically directed toward the association of HSA levels and CVD risk, the advances in the field of mass spectrometry research allow also a better characterization of the effects of oxidative modifications that could alter not only the structure but also the function of HSA.
... Another oxidative modification of proteins is tyrosine nitration, which is mainly generated through the oxidation of tyrosine by peroxynitrite and peroxynitrite-derived radicals or by the myeloperoxidase activity. Tyrosine nitration is highly selective (relatively few specific proteins are preferential targets) [80] and can cause dramatic changes in protein structure, inducing loss or gain of function as well as impairment of cellular processes relying on tyrosine phosphorylation cascades or protein turnover [81,82]. Many studies have suggested specific roles of 3-nitro-tyrosine in the functional changes related to disease conditions [82][83][84]. ...
... Tyrosine nitration is highly selective (relatively few specific proteins are preferential targets) [80] and can cause dramatic changes in protein structure, inducing loss or gain of function as well as impairment of cellular processes relying on tyrosine phosphorylation cascades or protein turnover [81,82]. Many studies have suggested specific roles of 3-nitro-tyrosine in the functional changes related to disease conditions [82][83][84]. Moreover, tyrosine nitration can induce autoimmune responses stimulating the production of specific antibodies against nitrated proteins [83]. ...
Under physio-pathological conditions, cells release membrane-surrounded structures named Extracellular Vesicles (EVs), which convey their molecular cargo to neighboring or distant cells influencing their metabolism. Besides their involvement in the intercellular communication, EVs might represent a tool used by cells to eliminate unnecessary/toxic material. Here, we revised the literature exploring the link between EVs and redox biology. The first proof of this link derives from evidence demonstrating that EVs from healthy cells protect target cells from oxidative insults through the transfer of antioxidants. Oxidative stress conditions influence the release and the molecular cargo of EVs that, in turn, modulate the redox status of target cells. Oxidative stress-related EVs exert both beneficial or harmful effects, as they can carry antioxidants or ROS-generating enzymes and oxidized molecules. As mediators of cell-to-cell communication, EVs are also implicated in the pathophysiology of oxidative stress-related diseases. The review found evidence that numerous studies speculated on the role of EVs in redox signaling and oxidative stress-related pathologies, but few of them unraveled molecular mechanisms behind this complex link. Thus, the purpose of this review is to report and discuss this evidence, highlighting that the analysis of the molecular content of oxidative stress-released EVs (reminiscent of the redox status of originating cells), is a starting point for the use of EVs as diagnostic and therapeutic tools in oxidative stress-related diseases.
... Nitrated αSyn holds promise as a biomarker for PD because it is a component of proteinaceous aggregates or Lewy inclusions [9,10]. Excess amount of nitrated αSyn is recognized as a salient feature of α-synucleinopathies [9,10,19,24,25] and is thought to be neurotoxic and accelerate αSyn aggregation [17,25]. Excess amount of nitrated αSyn is associated with nitrative stress, a type of oxidative stress that is characterized by nitrative modifications of proteins and other molecules due to an excess of nitric oxide and reactive nitrogen species. ...
... Nitrated αSyn holds promise as a biomarker for PD because it is a component of proteinaceous aggregates or Lewy inclusions [9,10]. Excess amount of nitrated αSyn is recognized as a salient feature of α-synucleinopathies [9,10,19,24,25] and is thought to be neurotoxic and accelerate αSyn aggregation [17,25]. Excess amount of nitrated αSyn is associated with nitrative stress, a type of oxidative stress that is characterized by nitrative modifications of proteins and other molecules due to an excess of nitric oxide and reactive nitrogen species. ...
Background. Salivary α-synuclein (aSyn) and its nitrated form, or 3-nitrotyrosine-α-synuclein (3-NT-αSyn), hold promise as biomarkers for idiopathic Parkinson’s disease (IPD). Nitrative stress that is characterized by an excess of 3-nitrotyrosine proteins (3-NT-proteins) has been proposed as a pathogenic mechanism in IPD. The objective is to study the pathological role of native αSyn, 3-NT-αSyn, and 3-NT-proteins in the saliva and submandibulary glands of patients with IPD. Methods. The salivary and serum αSyn and 3-NT-proteins concentration is evaluated with ELISA in patients and controls. Correlations of αSyn and 3-NT-proteins content with clinical features of the disease are examined. Immunohistochemical 3-NT-αSyn expression in submandibulary gland sections is analyzed. Results. (a) Salivary concentration and saliva/serum ratios of native αSyn and 3-NT-proteins are similar in patients and controls; (b) salivary αSyn and 3-NT-proteins do not correlate with any clinical feature; and (c) three patterns of 3-NT-αSyn-positive inclusions are observed on histological sections: rounded “Lewy-type” aggregates of 10–25 µm in diameter, coarse deposits with varied morphology, and spheroid inclusions or bodies of 3–5 µm in diameter. “Lewy-type” and coarse inclusions are observed in the interlobular connective tissue of the gland, and small-sized bodies are located within the cytoplasm of duct cells. “Lewy-type” inclusions are only observed in patients, and the remaining patterns of inclusions are observed in both the patients and controls. Conclusions. The patients’ saliva presents a similar concentration of native αSyn and 3-nitrotyrosine-proteins than that of the controls, and no correlations with clinical features are found. These findings preclude the utility of native αSyn in the saliva as a biomarker, and they indicate the absence of nitrative stress in the saliva and serum of patients. As regards nitrated αSyn, “Lewy-type” inclusions expressing 3-NT-αSyn are observed in the patients, not the controls—a novel finding that suggests that a biopsy of the submandibulary gland, if proven safe, could be a useful technique for diagnosing IPD. Finally, to our knowledge, this is also the first description of 3-NT-αSyn-immunoreactive intracytoplasmic bodies in cells that are located outside the nervous system. These intracytoplasmic bodies are present in duct cells of submandibulary gland sections from all subjects regardless of their pathology, and they can represent an aging or involutional change. Further immunostaining studies with different antibodies and larger samples are needed to validate the data.
... 13 In contrast, at elevated, micromolar concentrations, ˙NO employs non-traditional, indirect signaling mechanisms independent of cGMP. 14,15 This mode of action involves covalent post-translational modifications of proteins, notably S-nitrosylation, S-glutathionylation, and tyrosine nitration. Such non-traditional signaling arises from ˙NO interactions with prevalent intracellular reactants like O2 or O2•−, leading to the generation of various RNS (like nitrogen dioxide, ˙NO2; peroxynitrite, ONOO−; nitroxyl, HNO; dinitrogen trioxide, N2O3; and dinitrogen tetroxide, N2O4). ...
Reactive Nitrogen Species (RNS) and Reactive Sulphur Species (RSS) play crucial roles in numerous physiological pathways, including cellular signaling, metabolic cascades, and redox balance maintenance. In male reproduction, these molecules serve dual purposes: they support essential physiological functions but can cause harm if unregulated. This review explores the roles of RNS and RSS in male reproduction, emphasizing their beneficial and detrimental impacts. Physiologically, RNS and RSS aid in sperm maturation, capacitation, and initiating the acrosome reaction. Regulated synthesis of these species is vital for redox-regulated events essential for optimal male reproductive outcomes. Specifically, nitric oxide, a primary RNS, regulates sperm functions and penile erection mechanisms. However, imbalances, leading to excessive RNS and RSS levels, can cause oxidative and nitrosative stresses, which can trigger lipid peroxidation, protein alterations, and sperm DNA fragmentation, affecting sperm vitality, motility, and genomic integrity.
... In a scenario of oxidative stress, the reaction between superoxide anion and nitric oxide (NO) results in the formation of peroxynitrite. 3-NT generation can bring about conformational changes in proteins leading to permanent structural and functional damages which end up with damage to cells (Souza et al., 2008). It has now been established that 3-NT is a reliable marker indicating chronic immune system activation as well as neuronal death brought about by oxidative protein (Meredith et al., 2014). ...
The neurophysiological basis of autism spectrum disorder (ASD) is still uncertain. Nevertheless, studies support the hypotheses that oxidative stress, neuroinflammation, immune dysregulation, and metabolic stress are contributors. In this study, the serum levels of 3-nitrotyrosine (3-NT), hypoxia-inducible factor 1 α (HIF-1 α), heat shock protein 70 (HSP-70), interleukin-17A (IL-17A), IL-35, vitamin D3 (VITD), glucose transporter-1 (GUT1), and AMP-activated protein kinase (AMPK) were estimated in Saudi ASD children versus age-matched neurotypical controls, aiming to investigate whether these parameters have potential roles in the pathophysiologic mechanisms of ASD and hoping to find a reliable marker for early ASD diagnosis. This study included 25 ASD children and 25 typically developing children (3–11 years old). The diagnosis of ASD cases was made based on the Autism Diagnostic Observation Schedule (ADOS) and the Statistical Manual of Mental Disorders (DSM-5). ASD subjects were commonly male and revealed an intelligence quotient (IQ) < 70.The results detected that ASD children have remarkable greater serum levels of nitrosative stress (3-NT), hypoxia (HIF-1 α), inflammatory (HSP-70, IL-17A, and AMPK) biomarkers and lower serum levels of anti-inflammatory (IL-35 and VITD) and metabolic stress (GUT-1) biomarkers versus age-matched controls (P ≤ 0.0001). Pearson's correlation study revealed that 3-NT was positively associated with HIF-1 α and HSP-70. HIF-1 α was also positively correlated with HSP-70. AMPK was positively associated with GUT-1, however, IL-17A was negatively correlated with IL-35 and VITD.
Limitation:No specific therapeuticdrugs were administered in this study, and further studies are required to confirm the role of the selected biomarkers in ASD managements.
Conclusion
Changes in concentrations of different biomarkers indicate that they are involved in oxidative stress, metabolic stress, immune dysregulation and ASD pathogenesis. Hence, these parameters can prove to be promising biomarkers as well as therapeutic targets for the timely diagnosis and treatment of ASD patients.
... Tyr-NO 2 is a stable marker of oxidative and nitrosative stress in inflammatory disorders. The formation of Tyr-NO 2 can be described by the following reactions (Souza et al. 2008) Despite the fact, that Tyr-NO 2 is a stable marker of oxidative/nitrative stress in inflammatory diseases, further studies are necessary to carry out before it can be routinely used. Plasma levels of Tyr-NO 2 sensitively reflect the health status of an organism following pharmacotherapy. ...
A physiological level of oxygen/nitrogen free radicals and non-radical reactive species (collectively known as ROS/RNS) is termed oxidative eustress or “good stress” and is characterized by low to mild levels of oxidants involved in the regulation of various biochemical transformations such as carboxylation, hydroxylation, peroxidation, or modulation of signal transduction pathways such as Nuclear factor-κB (NF-κB), Mitogen-activated protein kinase (MAPK) cascade, phosphoinositide-3-kinase, nuclear factor erythroid 2–related factor 2 (Nrf2) and other processes. Increased levels of ROS/RNS, generated from both endogenous (mitochondria, NADPH oxidases) and/or exogenous sources (radiation, certain drugs, foods, cigarette smoking, pollution) result in a harmful condition termed oxidative stress (“bad stress”). Although it is widely accepted, that many chronic diseases are multifactorial in origin, they share oxidative stress as a common denominator. Here we review the importance of oxidative stress and the mechanisms through which oxidative stress contributes to the pathological states of an organism. Attention is focused on the chemistry of ROS and RNS (e.g. superoxide radical, hydrogen peroxide, hydroxyl radicals, peroxyl radicals, nitric oxide, peroxynitrite), and their role in oxidative damage of DNA, proteins, and membrane lipids. Quantitative and qualitative assessment of oxidative stress biomarkers is also discussed. Oxidative stress contributes to the pathology of cancer, cardiovascular diseases, diabetes, neurological disorders (Alzheimer’s and Parkinson’s diseases, Down syndrome), psychiatric diseases (depression, schizophrenia, bipolar disorder), renal disease, lung disease (chronic pulmonary obstruction, lung cancer), and aging. The concerted action of antioxidants to ameliorate the harmful effect of oxidative stress is achieved by antioxidant enzymes (Superoxide dismutases-SODs, catalase, glutathione peroxidase-GPx), and small molecular weight antioxidants (vitamins C and E, flavonoids, carotenoids, melatonin, ergothioneine, and others). Perhaps one of the most effective low molecular weight antioxidants is vitamin E, the first line of defense against the peroxidation of lipids. A promising approach appears to be the use of certain antioxidants (e.g. flavonoids), showing weak prooxidant properties that may boost cellular antioxidant systems and thus act as preventive anticancer agents. Redox metal-based enzyme mimetic compounds as potential pharmaceutical interventions and sirtuins as promising therapeutic targets for age-related diseases and anti-aging strategies are discussed.
... This protein nitration induces nitrative stress elevating the production of some specific biomarkers. Many studies suggest a strong correlation between 3-nitrotyrosine protein (NTP) and the development of various diseases, such as inflammatory conditions, neurodegenerative disease, etc. (Souza et al., 2008). Thus, NTP is a very promising biomarker of protein nitration by free peroxynitrite molecules (Bandookwala et al., 2020). ...
Extensive agricultural activities to feed the growing population are one major driving force behind aquatic pollution. Different types of pesticides are used in farmlands to increase crop production and wash up into water bodies. Glyphosate-based herbicide Roundup® is one of the most used pesticides in the United States; however, its effects on teleost species are still poorly understood. This study focused on the effects of environmentally relevant concentrations of Roundup exposure (low- and high-dose: 0.5 and 5 μg/L for 2-week) on Na+/K+-ATPase (NKA, a biomarker for sodium‑potassium ion pump efficacy), cytochrome P450-1A (CYP1A, a monooxygenase enzyme), 2,4-dinitrophenyl protein (DNP, a biomarker for protein oxidation), 3-nitrotyrosine protein (NTP, a biomarker for protein nitration), superoxidase dismutase (SOD, an antioxidant enzyme), catalase (CAT, an antioxidant enzyme) expressions, and cellular apoptosis in the gills of goldfish. Histopathological and in situ TUNEL analyses showed widespread tissue damage, including lamellar fusion, loss of gill architecture, club shape of primary lamellae, mucous formation, and distortion in the epithelium layer, as well as apoptotic nuclei in gills. Immunohistochemical and qRT-PCR analyses provided insights into the expressions of molecular indicators in gills. Fish exposed to Roundup exhibited a significant (P < 0.05) downregulation of NKA expression in gills. Additionally, we observed upregulation of CYP1A, DNP, NTP, SOD, and CAT expressions in the gills of goldfish. Overall, our results suggest that exposure to Roundup causes disruption of gill architecture, induces protein oxidation/nitration and cellular apoptosis, and alters prooxidant-antioxidant homeostasis in tissues, which may lead to reduced fitness and survivability of teleost species.
... In animal models, NO donors have been shown to reduce I/R injury [13,14]. However, exogenous NO donor administration has potentially deleterious effects; for example, NO has been shown to play a key role in apoptosis by reacting with superoxide to form peroxynitrite, inducing tyrosine nitration and deleterious protein changes [15,16]. There is also evidence that treatment with most organic nitrates at clinical doses impairs responsiveness to stimuli of endothelialderived NO release [17]. ...
Background
Decreased bioavailability of nitric oxide (NO) under hypoxic conditions can lead to endothelial dysfunction. NO supplementation may protect endothelial function in ischemia-reperfusion (IR) injury. Therefore, a meta-analysis of randomized controlled trials (RCTs) was performed to verify the protective effect of NO donors on endothelium in IR injury.
Methods
Medline, Embase, Cochrane Library, and Web of Science databases were searched from inception to April 1, 2023. The specific inclusion criteria were as follows: (1) RCTs; (2) trials comparing NO donors with placebo control groups; and (3) trials reporting the effects of these interventions on vascular endothelial functional outcomes in IR injury. Random-effects models were used to assess pooled effect sizes, which were expressed as standardized mean differences (SMD).
Results
Seven studies satisfied the inclusion criteria and consisted of a total of 149 participants. NO donors were protective of endothelial function in IR injury (SMD: − 1.60; 95% confidence interval [CI]: − 2.33, − 0.88, P < 0.0001; heterogeneity [I² = 66%, P = 0.001]). Results of the subgroup analysis showed the following: absence of protective effect of NO donor use following ischemia on endothelial function in IR injury − 1.78 (95% CI: − 2.50, − 1.07) and loss of protective effect on endothelial function after prolonged NO donor use − 0.89 (95% CI: − 2.06, 0.28).
Conclusion
The short-period use of NO donors before the onset of ischemia can protect endothelial function in IR injury.
... Its presence is indirect evidence of peroxynitrite synthesis and indicates oxidative stress [55]. Oxidative stress occurs when concentrations of ROS increase, and, knowing that Content courtesy of Springer Nature, terms of use apply. ...
Stress-induced neuroinflammation is a hallmark of modern society and has been linked to various emotional disorders, including anxiety. However, how microglia-associated neuroinflammation under chronic unpredictable mild stress (CUMS) alters mitochondrial function and subsequent medial prefrontal cortex-hippocampus (mPFC-HIPP) connectivity remains obscure. We speculated that CUMS might induce neuroinflammation, which involves altered mitochondrial protein levels, blockade of neuroinflammation by a microglial modulator, minocycline, protects against CUMS-induced alterations. Mice were exposed to CUMS for 3 weeks and received minocycline (50 mg/kg) intraperitoneally for 7 consecutive days during the 3rd week of CUMS. Novelty-suppressed feeding test and contextual anxiety test assessed anxiety-like behavior. Western blotting and immunofluorescent staining were employed to evaluate levels of proteins involved in neuroinflammation and mitochondrial function. In vivo dual-site extracellular recordings of local field potential (LFP) were conducted to evaluate the oscillatory activity and brain connectivity in mPFC-HIPP circuitry. We show that CUMS results in excessive microglial activation accompanied by aberrant levels of mitochondrial proteins, such as ATP-5A and the fission protein, Drp-1, increased oxidative stress indicated by elevated levels of nitrotyrosine, and decreased Nrf-2 levels. Furthermore, CUMS causes downregulation of α1 subunit of GABAAR, vesicular GABA transporter (Vgat), and glutamine synthetase (GS), leading to impaired LFP and connectivity of the mPFC-HIPP circuitry. Strikingly, blockage of microglial activation by minocycline ameliorates CUMS-induced aberrant levels of mitochondrial and GABAergic signaling proteins and prevents CUMS-induced anxiety-like behavior in mice. To the end, the study revealed that microglia is critically involved in stress-induced neuroinflammation, which may underlie the molecular mechanism of CUMS-induced anxiety behavior.
... to generate the satellite cell activation signal [37,63] as supported by the 3D-structure analysis of a HGF-c-met complex [64] (see Fig. S3 panel A for better understanding) , it appears reasonable to propose that nitrated Y198 is the specific location of the nitration-induced dysfunction of HGF/NK1 that disrupts activation of quiescent satellite cells by triggering conformational changes that alter the function and activity of the HGF/NK1 protein. Nitration disturbs protein function by a variety of mechanisms, and the direct disabling of nitrated-protein binding with its cognate receptor may be one of those mechanisms [65,66]. Conversely, loss of protein function requires a chemical modification at specific critical tyrosine residues. ...
Protein tyrosine residue (Y) nitration, a post-translational chemical-modification mode, has been associated with changes in protein activity and function; hence the accumulation of specific nitrated proteins in tissues may be used to monitor the onset and progression of pathological disorders. To verify the possible impact of nitration on postnatal muscle growth and regeneration, a pilot study was designed to examine the nitration/dysfunction of hepatocyte growth factor (HGF), a key ligand that is released from the extracellular tethering and activates myogenic stem satellite cells to enter the cell cycle upon muscle stretch and injury. Exposure of recombinant HGF (a hetero-dimer of α- and β-chains) to peroxynitrite induces Y nitration in HGF α-chain under physiological conditions. Physiological significance of this finding was emphasized by Western blotting that showed the NK1 segment of HGF (including a K1 domain critical for signaling-receptor c-met binding) undergoes nitration with a primary target of Y198. Peroxynitrite treatment abolished HGF-agonistic activity of the NK1 segment, as revealed by in vitro c-met binding and bromodeoxyuridine-incorporation assays. Importantly, direct-immunofluorescence microscopy of rat lower hind-limb muscles from two aged-groups (2-month-old "young" and 12-month-old "retired/adult") provided in vivo evidence for age-related nitration of extracellular HGF (Y198). Overall, findings provide the insight that HGF/NK1 nitration/dysfunction perturbs myogenic stem cell dynamics and homeostasis; hence NK1 nitration may stimulate progression of muscular disorders and diseases including sarcopenia.
... to generate the satellite cell activation signal [37,63] as supported by the 3D-structure analysis of a HGF-c-met complex [64] (see Fig. S3 panel A for better understanding) , it appears reasonable to propose that nitrated Y198 is the specific location of the nitration-induced dysfunction of HGF/NK1 that disrupts activation of quiescent satellite cells by triggering conformational changes that alter the function and activity of the HGF/NK1 protein. Nitration disturbs protein function by a variety of mechanisms, and the direct disabling of nitrated-protein binding with its cognate receptor may be one of those mechanisms [65,66]. Conversely, loss of protein function requires a chemical modification at specific critical tyrosine residues. ...
... Results indicated that these particular modifications did not alter the rate of degradation compared with native cyt c (155). Different is the case of CuZnSOD nitrated at Y108, where the modification enhances by twofold protein degradation by the proteosome (156), and thus the consequences of nitration of a single tyrosine in a protein may render different susceptibilities for proteosome degradation depending on the particular protein (157). For tyrosine-nitrated peptides the results coincide and show that the chymotrypsin activity of the 20S proteosome is largely inhibited (155,156) and this may render an incomplete proteolysis of the nitrated protein altering cell proteostasis. ...
The free radical nitric oxide (·NO) is a key mediator in different physiological processes such as vasodilation, neurotransmission, inflammation, and cellular immune responses, and thus preserving its bioavailability is essential. In several disease conditions, superoxide radical (O2·-) production increases and leads to the rapid "inactivation" of ·NO by a diffusion-controlled radical termination reaction that yields a potent and short-lived oxidant, peroxynitrite. This reaction not only limits ·NO bioavailability for physiological signal transduction but also can divert and switch the biochemistry of ·NO toward nitrooxidative processes. Indeed, since the early 1990s peroxynitrite (and its secondary derived species) has been linked to the establishment and progression of different acute and chronic human diseases and also to the normal aging process. Here, we revisit an earlier and classical review on the role of peroxynitrite in human physiology and pathology (Pacher P, Beckman J, Liaudet L. Physiol Rev 87: 315-424, 2007) and further integrate, update, and interpret the accumulated evidence over 30 years of research. Innovative tools and approaches for the detection, quantitation, and sub- or extracellular mapping of peroxynitrite and its secondary products (e.g., protein 3-nitrotyrosine) have allowed us to unambiguously connect the complex biochemistry of peroxynitrite with numerous biological outcomes at the physiological and pathological levels. Furthermore, our current knowledge of the ·NO/O2·- and peroxynitrite interplay at the cell, tissue, and organ levels is assisting in the discovery of therapeutic interventions for a variety of human diseases.
... The oxPTM 3-nitrotyrosine (nitroTyr, nY) is produced by the formation of peroxynitrite-derived radicals and their subsequent reaction with tyrosine side chains (Souza et al., 2008). Its accumulation in over 100 distinct proteins is associated with numerous disease pathologies (Pacher et al., 2007). ...
A critical step in developing therapeutics for oxidative stress-related pathologies is the ability to determine which specific modified protein species are innocuous by-products of pathology and which are causative agents. To achieve this goal, technologies are needed that can identify, characterize and quantify oxidative post translational modifications (oxPTMs). Nanobodies (Nbs) represent exquisite tools for intracellular tracking of molecules due to their small size, stability and engineerability. Here, we demonstrate that it is possible to develop a selective Nb against an oxPTM protein, with the key advance being the use of genetic code expansion (GCE) to provide an efficient source of the large quantities of high-quality, homogenous and site-specific oxPTM-containing protein needed for the Nb selection process. In this proof-of-concept study, we produce a Nb selective for a 3-nitrotyrosine (nitroTyr) modified form of the 14-3-3 signaling protein with a lesser recognition of nitroTyr in other protein contexts. This advance opens the door to the GCE-facilitated development of other anti-PTM Nbs.
... We suggest that this also takes place in other enzymes in which αKG is a co-factor in the inner mitochondrial membrane, as well as in the cytosol. αKG is the obligate co-substrate of Fe(II)/2-oxoglutarate-dependent dioxygenases (OGDD), a superfamily of enzymes that catalyse the oxidative decarboxylation of αKG, producing succinate and CO2 from O2 [20]. Prolyl hydroxylation of hypoxia-inducible factor (HIF)-α, as catalysed by the Fe(II)/2-oxoglutarate (AKG)-dependent prolyl hydroxylase domain (PHD) enzymes, has a hypoxia-sensing role in animals [21]. ...
The generation of peroxynitrite (ONOO −) is associated with several diseases, including atherosclerosis, hypertension, neurodegeneration, cancer, inflammation, and sepsis. Alpha-ke-toglutarate (αKG) is a known potential highly antioxidative agent for radical oxidative species such as peroxides. The question arises as to whether αKG is also a potential scavenger of ONOO − and a potential protector against ONOO −-mediated nitration of proteins. NMR studies of 1 mM αKG in 100 mM phosphate-buffered saline at pH 7.4 and pH 6.0 were carried out in the presence or absence of a final concentration of 2 mM ONOO −. An ONOO −-luminol-induced chemiluminescence reaction was used to measure the scavenging function of several concentrations of αKG; quantification of αKG was performed via spectrophotometric enzymatic assay of αKG in the absence or presence of 0, 1, or 2 mM ONOO −. The nitration of tyrosine residues on proteins was measured on ONOO −-treated bovine serum albumin (BSA) in the presence or absence of 0-24 mM αKG by an ELISA technique using a specific anti-IgG against nitro-tyrosine. The addition of ONOO − to αKG led to the formation of succinic acid and nitrite at pH 7.0, but not at pH 6.0, as αKG was stable against ONOO −. The absorbance of enzymatically estimated αKG at the time point of 30 min was significantly lower in favour of ONOO − (1 mM: 0.21 ± 0.03, 2 mM: 0.12 ± 0.05 vs. 0 mM: 0.32 ± 0.02; p < 0.001). The luminol technique showed an inverse logarithmic correlation of the ONOO − and αKG concentrations (y = −2 × 10 5 ln(x) + 1 × 10 6 ; r 2 = 0.99). The usage of 4 mM αKG showed a significant reduction by nearly half in the chemiluminescence signal (284,456 ± 29,293 cps, p < 0.001) compared to the control (474,401 ± 18,259); for 20 and 200 mM αKG, there were further reductions to 163,546 ± 26,196 cps (p < 0.001) and 12,658 ± 1928 cps (p < 0.001). Nitrated tyrosine residues were estimated using the ELISA technique. A negative linear correlation was obtained by estimating nitrated tyrosine residues in the presence of αKG (r 2 = 0.94): a reduction by half of nitrated tyrosine was estimated using 12 mM αKG compared to the control (326.1 ± 39.6 nmol vs. 844.5 ± 128.4 nmol; p < 0.001). Keywords: alpha-ketoglutarate (αKG); peroxynitrite (ONOO −); reactive oxygen and nitrogen species (RONS) Citation: Greilberger, J.; Greilberger, M.; Wintersteiger, R.; Zangger, K.; Herwig, R. Alpha-Ketoglutarate: A Potential Inner Mitochondrial and Cytosolic Protector against Peroxynitrite and Peroxynitrite-Induced Nitration? Antioxidants
... Nitration of protein tyrosine is a selective process despite the fact that no consensus sequence ensuring selectivity has been convincingly confirmed (Bartesaghi and Radi, 2018). Rather, some common features appear to affect YNO 2 formation such as the presence of acidic residues next to the YNO 2 site, cysteine or methionine neighbouring the target tyrosine residue and the presence of loop-forming amino acids such as proline or glycine (Souza et al., 2008). Beyond the amino acid sequence, additional factors influence the Table 1 List of software tools developed so far for predicting NO-dependent PTMs (S-nitrosation and tyrosine nitration). ...
The perception and transduction of nitric oxide (NO) signal is achieved by NO-dependent posttranslational modifications (PTMs) among which S-nitrosation and tyrosine nitration has biological significance. In plants, 100-1000 S-nitrosated and tyrosine nitrated proteins have been identified so far by mass spectrometry. The determination of NO-modified protein targets/amino acid residues is often methodologically challenging. In the past decade, the growing demand for the knowledge of S-nitrosated or tyrosine nitrated sites has motivated the introduction of bioinformatics tools. For predicting S-nitrosation seven computational tools have been developed (GPS-SNO, SNOSite, iSNO-PseACC, iSNO-AAPAir, PSNO, PreSNO, RecSNO). Four predictors have been developed for indicating tyrosine nitration sites (GPS-YNO2, iNitro-Tyr, PredNTS, iNitroY-Deep), and one tool (DeepNitro) predicts both NO-dependent PTMs. The advantage of these computational tools is the fast provision of large amount of information. In this review, the available software tools have been tested on plant proteins in which S-nitrosated or tyrosine nitrated sites have been experimentally identified. The predictors showed distinct performance and there were differences from the experimental results partly due to the fact that the three-dimensional protein structure is not taken into account by the computational tools. Nevertheless, the predictors excellently establish experiments, and it is suggested to apply all available tools on target proteins and compare their results. In the future, computational prediction must be developed further to improve the precision with which S-nitrosation/tyrosine nitration-sites are identified.
... In fact, nitrotyrosine detection is believed to be a biomarker of RNS generation and protein damages [54][55][56]. The protein-tyrosine-nitration can affect protein structure and function leading to macromolecules damages such as inactivation of certain enzymes including catalase and MnSOD [57,58]. Therefore, we can suggest that the loss of SOD and GPx activities in our study is caused by the increase of protein nitrotyrosination observed in 7β-OHC-treated cells. ...
Oxysterols are assumed to be the driving force behind numerous neurodegenerative diseases. In this work, we aimed to study the ability of 7β-hydroxycholesterol (7β-OHC) to trigger oxidative stress and cell death in human neuroblastoma cells (SH-SY5Y) then the capacity of Nigella sativa and Milk thistle seed oils (NSO and MTSO respectively) to oppose 7β-OHC-induced side effects. The impact of 7β-OHC, associated or not with NSO or MTSO, was studied on different criteria: cell viability; redox status and apoptosis. Oxidative stress was assessed through the intracellular reactive oxygen species (ROS) production, levels of enzymatic and non-enzymatic antioxidants, lipidand protein oxidation products. Our results indicate that 7β-OHC [40 µg/mL] exhibit pr-oxidative and pro-apoptotic activities shown by a decrease of the antioxidant enzymatic activities and an increase of ROS production, lipid, and protein oxidation end products as well as nitrotyrosine formation and caspase 3 activation. However, under the pre-treatment with NSO, and especially with MTSO [100 µg/mL], a marked attenuation of oxidative damages was observed. Our study suggests harmful effects of 7β-OHC consisting of pro-oxidative,anti-proliferativeand pro-apoptotic activities that may contribute to neurodegeneration. NSO and especially MTSO showed potential cytoprotection against the cytotoxicity of 7β-OHC.
... A large number of transcription factors are also affected by NO. essentially enzymatic (Seth et al., 2018). Similarly to S-nitrosylation, NO does not react directly with tyrosines but it can indirectly through the formation of peroxynitrite (Souza, Peluffo, & Radi, 2008). Tyrosine nitration has less capacity for enzymatic inhibition than S-nitrosylations and, additionally, requires the presence of reactive oxygen species. ...
Nitric oxide (NO) is a reactive gaseous molecule that has several functions in biological systems depending on its concentration. At low concentrations, NO acts as a signaling molecule, while at high concentrations, it becomes very toxic due to its ability to react with multiple cellular targets. Soil bacteria, commonly known as rhizobia, have the capacity to establish a N2-fixing symbiosis with legumes inducing the formation of nodules in their roots. Several reports have shown NO production in the nodules where this gas acts either as a signaling molecule which regulates gene expression, or as a potent inhibitor of nitrogenase and other plant and bacteria enzymes. A better understanding of the sinks and sources of NO in rhizobia is essential to protect symbiotic nitrogen fixation from nitrosative stress. In nodules, both the plant and the microsymbiont contribute to the production of NO. From the bacterial perspective, the main source of NO reported in rhizobia is the denitrification pathway that varies significantly depending on the species. In addition to denitrification, nitrate assimilation is emerging as a new source of NO in rhizobia. To control NO accumulation in the nodules, in addition to plant haemoglobins, bacteroids also contribute to NO detoxification through the expression of a NorBC-type nitric oxide reductase as well as rhizobial haemoglobins. In the present review, updated knowledge about the NO metabolism in legume-associated endosymbiotic bacteria is summarized.
... Tyrosine nitration is the addition of a nitro (-NO 2 ) group to the tyrosine residue of the target protein, which can promote conformational changes that can lead to gain, loss, or no change in the function of the target protein (Kolbert et al., 2017). Tyrosine nitration induces structural changes by enhancing the hydrophobicity of tyrosine residues (Souza et al., 2008). The pea APX1 was shown to have two of its tyrosines nitrated, of which Tyr235 has been suggested to be the most eligible candidate for inhibiting the activity of APX as it is present close to the pocket of the catalytic center and 3.6 Å away from the heme group (Patterson et al., 1995;Mandelman et al., 1998;Begara-Morales et al., 2014). ...
Ascorbate peroxidase (APX) is an important reactive oxygen species (ROS)-scavenging enzyme, which catalyzes the removal of hydrogen peroxide (H2O2) to prevent oxidative damage. The peroxidase activity of APX is regulated by posttranslational modifications (PTMs), such as S-nitrosylation, tyrosine nitration, and S-sulfhydration. In addition, it has been recently reported that APX functions as a molecular chaperone, protecting rice against heat stress. In this study, we attempted to identify the various functions of APX in Arabidopsis and the effects of PTMs on these functions. Cytosol type APX1 from Arabidopsis thaliana (AtAPX1) exists in multimeric forms ranging from dimeric to high-molecular-weight (HMW) complexes. Similar to the rice APX2, AtAPX1 plays a dual role behaving both as a regular peroxidase and a chaperone molecule. The dual activity of AtAPX1 was strongly related to its structural status. The main dimeric form of the AtAPX1 protein showed the highest peroxidase activity, whereas the HMW form exhibited the highest chaperone activity. Moreover, in vivo studies indicated that the structure of AtAPX1 was regulated by heat and salt stresses, with both involved in the association and dissociation of complexes, respectively. Additionally, we investigated the effects of S-nitrosylation, S-sulfhydration, and tyrosine nitration on the protein structure and functions using gel analysis and enzymatic activity assays. S-nitrosylation and S-sulfhydration positively regulated the peroxidase activity, whereas tyrosine nitration had a negative impact. However, no effects were observed on the chaperone function and the oligomeric status of AtAPX1. Our results will facilitate the understanding of the role and regulation of APX under abiotic stress and posttranslational modifications.
... Nitration of protein occurs basically through the metabolism of nitric oxide by the production of some metabolites, which include nitrogen dioxide (NO 2 ) and peroxynitrite (ONOO − ) (Griffiths et al., 2006). Amino acids can be nitrated by the action of ONOO − , and this is seen in its reaction with tyrosine which ultimately causes a conformational change (Souza et al., 2008). Various aspects of protein function can thus be regulated by this modification in proteins. ...
... Reduction in 3-Nitrotyrosine levels is indicative of oxidative stress-related brain and central nervous system pathologies. 2 Korde Ch et al. [14] 2013 Cancer Related Events NitroY is a nitrosative biomarker showing overproduction of NO, which has been suggested to modulate different cancer-related events including angiogenesis, apoptosis, cell cycle, invasion, and metastasis. 3 Gochman et al. [15] 2012 Colon Cancer iNOS expression and NitroY may be an indicator of cancer development and progression in inflammation and colon cancer in humans. ...
In biological systems, Nitration is a crucial post-translational modification which occurs on various amino acids. Nitration of Tyrosine is regarded as nitorsative stress biomarker resulting in the formation of peroxynitrite and other reactive and harmful nitrogen species. NitroTyrosine is closely related to Carcinogenesis, tumor growth progression and other major pathological conditions including systemic autoimmune diseases, inflammation, neurodegeneration and cardiovascular disorders. Additionally, the alteration in Nitrotyrosine profile occurs well before appearance of any symptoms of aforementioned diseases making nitrotyrosine a biomarker and potential target for early prognosis of aforementioned diseases. The wet lab identification of potential nitrotyrosine sites is laborious, time-taking and costly due to challenges of in vitro, ex vivo and in vivo identification processes. To supplement wet lab identification of nitrotyrosine, we proposed, implemented and evaluated a different approach to develop tyrosine nitration site predictors using pseudo amino acid compositions (PseAAC) and deep neural networks (DNNs). Proposed approach does not require any feature extraction and uses DNNs for learning a feature representation of peptide sequences and classification thereof. Validation of proposed approach is done using well-known model evaluation measures. Among different deep neural networks, convolutional neural network-based predictor achieved best scores on independent dataset with accuracy of 87.2%, matthew’s correlation coefficient score of 0.74 and AuC score of 0.91 which outperforms the previous reported scores of Nitrotyrosine predictors.
... Oxidative stress (OS) is a state of disturbed balance between oxidation and reduction processes. There is an overproduction of reactive oxygen and nitrogen species (ROS/RNS), which causes structural changes and damage to lipid molecules, nucleic acids and proteins, resulting in modification of their biological functions or complete deactivation [1][2][3][4][5]. ...
Oxidative stress (OS) in the foetal and neonatal periods leads to many disorders in newborns and in later life. The nutritional status of pregnant women is considered to be one of the key factors that triggers OS. We investigated the relationship between the concentration of selected mineral elements in the blood of pregnant women and the concentration of 3′nitrotyrosine (3′NT) as a marker of OS in the umbilical cord blood of newborns. The study group consisted of 57 pregnant women and their newborn children. The concentrations of magnesium (Mg), calcium (Ca), iron (Fe), zinc (Zn) and copper (Cu) in maternal serum (MS) were measured by the flame atomic absorption/emission spectrometry (FAAS/FAES) method. The concentration of 3′NT in umbilical cord serum (UCS) of newborns was determined by the ELISA method. A positive correlation between MS Fe and UCS 3′NT in male newborns was shown (rho = 0.392, p = 0.053). Significantly higher UCS 3′NT was demonstrated in newborns, especially males, whose mothers were characterized by MS Fe higher than 400 μg/dL compared to those of mothers with MS Fe up to 300 μg/dL (p < 0.01). Moreover, a negative correlation between the MS Cu and UCS 3′NT in male newborns was observed (rho = −0.509, p = 0.008). Results of the study showed the need to develop strategies to optimize the nutritional status of pregnant women. Implementation of these strategies could contribute to reducing the risk of pre- and neonatal OS and its adverse health effects in the offspring.
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Protein tyrosine nitration is a selectively and reversible important post-translational modification, which is closely related to oxidative stress. Astrocytoma is the most common neuroepithelial tumor with heterogeneity and complexity. In the past, the diagnosis of astrocytoma was based on the histological and clinical features, and the treatment methods were nothing more than surgery-assisted radiotherapy and chemotherapy. Obviously, traditional methods short falls an effective treatment for astrocytoma. In late 2021, the World Health Organization (WHO) adopted molecular biomarkers in the comprehensive diagnosis of astrocytoma, such as IDH-mutant and DNA methylation, which enabled the risk stratification, classification, and clinical prognosis prediction of astrocytoma to be more correct. Protein tyrosine nitration is closely related to the pathogenesis of astrocytoma. We hypothesize that nitroproteome is significantly different in astrocytoma relative to controls, which leads to establishment of nitroprotein biomarkers for patient stratification, diagnostics, and prediction of disease stages and severity grade, targeted prevention in secondary care, treatment algorithms tailored to individualized patient profile in the framework of predictive, preventive, and personalized medicine (PPPM; 3P medicine). Nitroproteomics based on gel electrophoresis and tandem mass spectrometry is an effective tool to identify the nitroproteins and effective biomarkers in human astrocytomas, clarifying the biological roles of oxidative/nitrative stress in the pathophysiology of astrocytomas, functional characteristics of nitroproteins in astrocytomas, nitration-mediated signal pathway network, and early diagnosis and treatment of astrocytomas. The results finds that these nitroproteins are enriched in mitotic cell components, which are related to transcription regulation, signal transduction, controlling subcellular organelle events, cell perception, maintaining cell homeostasis, and immune activity. Eleven statistically significant signal pathways are identified in astrocytoma, including remodeling of epithelial adherens junctions, germ cell-sertoli cell junction signaling, 14-3-3-mediated signaling, phagosome maturation, gap junction signaling, axonal guidance signaling, assembly of RNA polymerase III complex, and TREM1 signaling. Furthermore, protein tyrosine nitration is closely associated with the therapeutic effects of protein drugs, and molecular mechanism and drug targets of cancer. It provides valuable data for studying the protein nitration biomarkers, molecular mechanisms, and therapeutic targets of astrocytoma towards PPPM (3P medicine) practice.
Supplementary information:
The online version contains supplementary material available at 10.1007/s13167-023-00348-y.
Reactive oxygen species (ROS) are produced during oxidative metabolism in aerobic organisms. Under normal conditions, ROS production and elimination are in a relatively balanced state. However, under internal or external environmental stress, such as high glucose levels or UV radiation, ROS production can increase significantly, leading to oxidative stress. Excess ROS production not only damages biomolecules but is also closely associated with the pathogenesis of many diseases, such as skin photoaging, diabetes, and cancer. Antioxidant peptides (AOPs) are naturally occurring or artificially designed peptides that can reduce the levels of ROS and other pro-oxidants, thus showing great potential in the treatment of oxidative stress-related diseases. In this review, we discussed ROS production and its role in inducing oxidative stress-related diseases in humans. Additionally, we discussed the sources, mechanism of action, and evaluation methods of AOPs and provided directions for future studies on AOPs.
Peroxynitrite (ONOO−/ONOOH) is a strong oxidant formed in vivo. It is cytotoxic at high concentrations, participates in different diseases and the aging process, and contributes to immune cell response mechanisms to invading organisms. The mechanism of action of peroxynitrite in these processes depends on the oxidation of biomolecules either by direct reaction or by the reaction of derived secondary species. This review presents the biological chemistry of peroxynitrite and the derived radicals nitrogen dioxide, hydroxyl radical and carbonate radical, particularly discussing their diffusion properties across cellular membranes and in the context of their short biological half-lives. While in some cases simple diffusion is the main route, peroxynitrite can also use anion channels to traverse cellular membranes. Furthermore, the potential role of aquaporins in facilitating the diffusion of peroxynitrite and its neutral conjugate acid is discussed. The wide range of membrane permeabilities exhibited by precursors, peroxynitrite and its secondary radical species results in an unequal compartmentalization of peroxynitrite formation and reactions and decay, thus partially defining the biological sites of action of peroxynitrite.
A keratotic plug (KP) is a solid mass primarily composed of protein that forms in pores, and preventing their formation has been challenging. The presence of keratin 17 (K17), a characteristic of KPs, implies the involvement of interferon-gamma (IFN-γ), which contributes to K17 formation, in KP formation. IFN-γ is also involved in the production of nitric oxide (NO), and NO-mediated nitration of tyrosine is known to induce protein aggregation, which is one of the possible mechanisms by which proteins become solids. In this study, to clarify the involvement of IFN-γ and protein nitration in KPs, we investigated the relationship between the amount of IFN-γ collected from the skin around nasal alae and the number of KPs, protein components of KPs, and the effect of nitration on molecular sizes of proteins in vitro. As a result, more KPs were detected in individuals with high IFN-γ levels, and nitrotyrosine was present within the KPs. Protein nitration increased the molecular size of proteins, and IFN-γ increased nitrotyrosine production in keratinocytes in the same manner as in NO donors. Collectively, these findings indicate that IFN-γ-mediated protein nitration via NO generation causes solid mass formation in the pores. Inhibition of protein nitration followed by protein aggregation could be effective in preventing KP formation.
This expansive and practical textbook contains organic chemistry experiments for teaching in the laboratory at the undergraduate level covering a range of functional group transformations and key organic reactions.The editorial team have collected contributions from around the world and standardized them for publication. Each experiment will explore a modern chemistry scenario, such as: sustainable chemistry; application in the pharmaceutical industry; catalysis and material sciences, to name a few. All the experiments will be complemented with a set of questions to challenge the students and a section for the instructors, concerning the results obtained and advice on getting the best outcome from the experiment. A section covering practical aspects with tips and advice for the instructors, together with the results obtained in the laboratory by students, has been compiled for each experiment.
Targeted at professors and lecturers in chemistry, this useful text will provide up to date experiments putting the science into context for the students.
Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disease that causes progressive joint damage and can lead to lifelong disability. Numerous studies support the hypothesis that reactive oxygen species (ROS) are associated with RA pathogenesis. Recent advances have clarified the anti-inflammatory effect of antioxidants and their roles in RA alleviation. In addition, several important signaling pathway components, such as nuclear factor kappa B, activator-protein-1, nuclear factor (erythroid-derived 2)-like 2/kelch-like associated protein, signal transducer and activator of transcription 3, and mitogen-activated protein kinases, including c-Jun N-terminal kinase, have been identified to be associated with RA. In this paper, we outline the ROS generation process and relevant oxidative markers, thereby providing evidence of the association between oxidative stress and RA pathogenesis. Furthermore, we describe various therapeutic targets in several prominent signaling pathways for improving RA disease activity and its hyper oxidative state. Finally, we reviewed natural foods, phytochemicals, chemical compounds with antioxidant properties and the association of microbiota with RA pathogenesis.
Ribonucleotide reductase (RNR) is a multi-subunit enzyme responsible for catalyzing the rate-limiting step in the production of deoxyribonucleotides essential for DNA synthesis and repair. The active RNR complex is composed of multimeric R1 and R2 subunits. The RNR catalysis involves the formation of tyrosyl radicals in R2 subunits and thiyl radicals in R1 subunits. Despite the quarternary structure and cofactor diversity, all the three classes of RNR have a conserved cysteine residue at the active site which is converted into a thiyl radical that initiates the substrate turnover, suggesting that the catalytic mechanism is somewhat similar for all three classes of the RNR enzyme. Increased RNR activity has been associated with malignant transformation, cancer cell growth, and tumorigenesis. Efforts concerning the understanding of RNR inhibition in designing potent RNR inhibitors/drugs as well as developing novel approaches for antibacterial, antiviral treatments, and cancer therapeutics with improved radiosensitization have been made in clinical research. This review highlights the precise and potent roles of NO in RNR inhibition by targeting both the subunits. Under nitrosative stress, the thiols of the R1 subunits have been found to be modified by S-nitrosylation and the tyrosyl radicals of the R2 subunits have been modified by nitration. In view of the recent advances and progresses in the field of nitrosative modifications and its fundamental role in signaling with implications in health and diseases, the present article focuses on the regulations of RNR activity by S-nitrosylation of thiols (R1 subunits) and nitration of tyrosyl residues (R2 subunits) which will further help in developing design new drugs and therapies.
In plants reactive oxygen species (ROS) generation under both normal as well as extreme/adverse conditions is a normal phenomenon; however, under extreme environmental conditions, production of ROS exceeds the capacity of the antioxidant defense mechanisms (enzymatic and nonenzymatic) that are key in ameliorating the deleterious impacts of ROS. The ascorbate–glutathione (AsA‐GSH) cycle plays an imperative role in ROS detoxification and protects plants from stress‐induced damage. Nitric oxide (NO) is considered as a signaling molecule and can crosstalk with several signaling molecules when present at optimal concentrations. Experimental evidence has shown that application of NO upregulates this cycle by influencing its mutually functioning enzymatic and nonenzymatic components. By regulating signaling, NO maintains the metabolic processes of plants to combat stressful conditions. Crosstalk of NO with the plant hormones and other endogenous molecules triggers several downstream signaling events thereby regulating the key cellular functions. The majority of plant hormones influence the endogenous synthesis of NO imparting significant influence on plant cellular activities. The influence of phytohormone–NO interactions on the AsA‐GSH pathway and stress tolerance has been reported. In this review our main focus is on the latest advancements in NO research and regulation of the AsA‐GSH pathway vis‐à‐vis the impact of phytohormones.
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are the mediators of redox activity and are known to perform concentration-specific bimodal roles. At lower concentrations, serves as a molecular messenger and signaling molecule while at higher concentrations induces stress which in turn alters the sperm's functional characteristics. Production of ROS and RNS cannot be prevented entirely and should not be followed as a pragmatic approach as they are involved in numerous sperm physiological functions. When the antioxidants defense armory is meager, excess generation of these species cross the physiological limits and inactivates essential metabolic enzymes and disrupts signal transduction altering normal sperm functions. As per the available literature, oxidants mostly arise as a result of pathological conditions or cryopreservation-induced injury. Dead and debilitated or abnormal spermatozoa and associated leukocytes release free radicals in an excess amount which elicits oxidative and nitrosative stressors that are potentially toxic to cryosurviving sperm. ROS plays a double edge sword effect on sperm function, as regulators of physiological mechanisms at low levels and as toxicants when produced at high concentrations. Recently nitric oxide (NO.) has emerged as a potential regulator of sperm physiology, in addition, found to mediate homeostasis of the seminal plasma microenvironment when semen samples are incubated with optimal concentrations of NO. compounds. The NO. compounds can provide some resistance to future stresses which are not usually harnessed by using the defensive strategy of supplementing antioxidants. Therefore, through the optimized addition of NO. donor and inhibitor in extender, the free radical-induced damage can be avoided without inhibiting their essential physiological effects on fertilization and subsequent embryo development. This article is intended to describe the role of reactive oxidants in the physiology and pathophysiology of spermatozoa and their relationship with various seminal attributes.
Preclinical studies reveal maternal exercise as a promising intervention to reduce the transmission of multi-generational metabolic dysfunction caused by maternal obesity. The benefits of maternal exercise on offspring health may arise from multiple factors and have recently been shown to involve DNA demethylation of critical hepatic genes leading to enhanced glucose metabolism in offspring. Histone modification is another epigenetic regulator, yet the effects of maternal obesity and exercise on histone methylation in offspring are not known. Here, we find that maternal high fat diet (HFD; 60% kcal from fat) induced dysregulation of offspring liver glucose metabolism in C57BL/6 mice through mechanism involving increased reactive oxygen species, WD repeat-containing 82 (WDR82) carbonylation, and inactivation of H3K4 methyltransferase leading to decreased H3K4me3 at the promoters of glucose metabolic genes. Remarkably, the entire signal was restored if the HFD-fed dams had exercised during pregnancy. WDR82 overexpression in hepatoblasts mimicked the effects of maternal exercise on H3K4me3 levels. Placental superoxide dismutase 3 (SOD3), but not antioxidant treatment with N-acetylcysteine was necessary for the regulation of H3K4me3, gene expression and glucose metabolism. Maternal exercise regulates a multi-component epigenetic system in fetal liver resulting in the transmission of the benefits of exercise to offspring.
Parkinson's disease is characterised by the presence in brain tissue of aberrant inclusions known as Lewy bodies and Lewy neurites, which are deposits composed by α-synuclein and a variety of other cellular components, including in particular lipid membranes. The dysregulation of the balance between lipid homeostasis and α-synuclein homeostasis is therefore likely to be closely involved in the onset and progression of Parkinson's disease and related synucleinopathies. As our understanding of this balance is increasing, we describe recent advances in the characterisation of the role of post-translational modifications in modulating the interactions of α-synuclein with lipid membranes. We then discuss the impact of these advances on the development of novel diagnostic and therapeutic tools for synucleinopathies.
Nitric oxide (•NO) is a free radical which exerts a myriad of functions in the physiology of higher plants either under physiological and environmental stress conditions. NO, and derived molecules designated as reactive nitrogen species (RNS), can mediate posttranslational modifications (PTMs) of proteins which can affect their functionality. Among these NO/RNS-derived PTMs, it can be highlighted S-nitrosation, metal nitrosylation and nitration. This last one involves the addition of a nitro group (-NO2) to some specific amino acids such as tyrosine or tryptophan. An increase in the content of protein nitration has been recognized as a suitable marker of nitro-oxidative stress which is frequently associated with oxidative stress under diverse environmental stress conditions. This mini-review aims to provide a comprehensive overview of protein nitration and its significance in higher plants.
Plants respond to the limited or excess supply of metalloids, boron (B), silicon (Si), selenium (Se), arsenic (As), and antimony (Sb) via complex signaling pathways that are mainly regulated by nitric oxide (NO). The absorption of metalloids from the soil is facilitated by pathways that involve aquaporins, aquaglyceroporins, phosphate, and sulfate transporters; however, their regulation by NO is poorly understood. Using in silico software, we predicted the S-nitrosation of known metalloid transporters, proposing NO-dependent regulation of metalloid transport systems at the posttranslational level. NO intensifies the stress-mitigating effect of Si, whereas in the case of Se, As, and Sb, the accumulation of NO or reactive nitrogen species contributes to toxicity. NO promotes the beneficial effect of low Se concentrations and mitigates the damage caused by B deficiency. In addition, the exogenous application of NO donor, sodium nitroprusside, reduces B, Se, and As toxicity. The primary role of NO in metalloid stress response is to mitigate oxidative stress by activating antioxidant defense at the level of protein activity and gene expression. This review discusses the role of NO in plant responses to metalloids and suggests future research directions.
Accumulation of the neuronal presynaptic protein alpha-synuclein within proteinaceous inclusions represents the key histophathological hallmark of a spectrum of neurodegenerative disorders, referred to by the umbrella term a-synucleinopathies. Even though alpha-synuclein is expressed predominantly in neurons, pathological aggregates of the protein are also found in the glial cells of the brain. In Parkinson’s disease and dementia with Lewy bodies, alpha-synuclein accumulates mainly in neurons forming the Lewy bodies and Lewy neurites, whereas in multiple system atrophy, the protein aggregates mostly in the glial cytoplasmic inclusions within oligodendrocytes. In addition, astrogliosis and microgliosis are found in the synucleinopathy brains, whereas both astrocytes and microglia internalize alpha-synuclein and contribute to the spread of pathology. The mechanisms underlying the pathological accumulation of alpha-synuclein in glial cells that under physiological conditions express low to non-detectable levels of the protein are an area of intense research. Undoubtedly, the presence of aggregated alpha-synuclein can disrupt glial function in general and can contribute to neurodegeneration through numerous pathways. Herein, we summarize the current knowledge on the role of alpha-synuclein in both neurons and glia, highlighting the contribution of the neuron-glia connectome in the disease initiation and progression, which may represent potential therapeutic target for a-synucleinopathies.
Background:
Neuropathological features of Alzheimer's disease are characterized by the deposition of amyloid-β (Aβ) plaques and impairments in synaptic activity and memory. However, we know little about the physiological role of amyloid-β protein precursor (AβPP) from which Aβ derives.
Objective:
Evaluate APP deficiency induced alterations in neuronal electrical activity and mitochondrial protein expression.
Methods:
Utilizing electrophysiological, biochemical, pharmacological, and behavioral tests, we revealed aberrant local field potential (LFP), extracellular neuronal firing and levels of mitochondrial proteins.
Result:
We show that APP knockout (APP -/- ) leads to increased gamma oscillations in the medial prefrontal cortex (mPFC) at 1-2 months old, which can be restored by baclofen (Bac), a γ-aminobutyric acid type B receptor (GABABR) agonist. A higher dose and longer exposure time is required for Bac to suppress neuronal firing in APP -/- mice than in wild type animals, indicating enhanced GABABR mediated activity in the mPFC of APP -/- mice. In line with increased GABABR function, the glutamine synthetase inhibitor, L-methionine sulfonate, significantly increases GABABR levels in the mPFC of APP -/- mice and this is associated with a significantly lower incidence of death. The results suggest that APP -/- mice developed stronger GABABR mediated inhibition. Using HEK 293 as an expression system, we uncover that AβPP functions to suppress GABABR expression. Furthermore, APP -/- mice show abnormal expression of several mitochondrial proteins.
Conclusion:
APP deficiency leads to both abnormal network activity involving defected GABABR and mitochondrial dysfunction, suggesting critical role of AβPP in synaptic and network function.
Cells are continually exposed to endogenous reactive oxygen, nitrogen, and halogen species, causing damage to biomolecules. Among them, peroxynitrite and hypochlorous acid are not only oxidants but also biological nitrating and chlorinating agents, leading to the formation of 3-nitrotyrosine and 3-chlorotyrosine, respectively, in proteins. 3-Nitrotyrosine has been detected in vivo under several pathophysiological conditions, including breast cancer. Studies show that the concentrations of 3-nitrotyrosine in plasma proteins and platelets were significantly elevated in breast cancer patients. Compared to blood serum albumin, hemoglobin adducts represent biomonitoring of exposure with a longer lifetime. In this study, human hemoglobin was freshly isolated from blood and digested into peptides with trypsin, and the levels of protein adducts, including nitration, nitrosylation, and chlorination of tyrosine as well as oxidation of methionine residues, were simultaneously quantified by nanoflow liquid chromatography nanoelectrospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) with selected reaction monitoring. The results demonstrated that the relative extents of nitration at α-Tyr-42 and β-Tyr-130, nitrosylation at α-Tyr-24, and chlorination at α-Tyr-24 and β-Tyr-130 are significantly higher in globin of 25 breast cancer patients compared to those in 25 healthy subjects (p < 0.05). In particular, nitration at α-Tyr-42 and chlorination at α-Tyr-24 showed the area under the receiver operating characteristic curve of >0.8. While the age of the subjects is correlated with the extents of some of these adducts, the body mass index does not have an effect on any of them. Starting with 1 drop of blood, our results indicated that this highly sensitive and specific nanoLC-NSI/MS/MS is useful in investigating the role of reactive nitrogen oxide species and reactive chlorine species in the etiology of breast cancer.
Eosinophil peroxidase (EPO) has been implicated in promoting oxidative tissue injury in conditions ranging from asthma and
other allergic inflammatory disorders to cancer and parasitic/helminthic infections. Studies thus far on this unique peroxidase
have primarily focused on its unusual substrate preference for bromide (Br−) and the pseudohalide thiocyanate (SCN−) forming potent hypohalous acids as cytotoxic oxidants. However, the ability of EPO to generate reactive nitrogen species
has not yet been reported. We now demonstrate that EPO readily uses nitrite (NO2
−), a major end-product of nitric oxide (⋅NO) metabolism, as substrate to generate a reactive intermediate that nitrates protein tyrosyl residues in high yield. EPO-catalyzed
nitration of tyrosine occurred more readily than bromination at neutral pH, plasma levels of halides, and pathophysiologically
relevant concentrations of NO2
−. Furthermore, EPO was significantly more effective than MPO at promoting tyrosine nitration in the presence of plasma levels
of halides. Whereas recent studies suggest that MPO can also promote protein nitration through indirect oxidation of NO2
− with HOCl, we found no evidence that EPO can indirectly mediate protein nitration by a similar reaction between HOBr and
NO2
−. EPO-dependent nitration of tyrosine was modulated over a physiologically relevant range of SCN− concentrations and was accompanied by formation of tyrosyl radical addition products (e.g. o,o′-dityrosine, pulcherosine, trityrosine). The potential role of specific antioxidants and nucleophilic scavengers on yields
of tyrosine nitration and bromination by EPO are examined. Thus, EPO may contribute to nitrotyrosine formation in inflammatory
conditions characterized by recruitment and activation of eosinophils.
Involvement of peroxynitrite (ONOO−) in inflammatory diseases has been implicated by detection of 3-nitrotyrosine, an allegedly characteristic protein oxidation
product, in various inflamed tissues. We show here that nitrite (NO2−), the primary metabolic end product of nitric oxide (NO·), can be oxidized by the heme peroxidases horseradish peroxidase, myeloperoxidase (MPO), and lactoperoxidase (LPO), in the
presence of hydrogen peroxide (H2O2), to most likely form NO·2, which can also contribute to tyrosine nitration during inflammatory processes. Phenolic nitration by MPO-catalyzed NO2− oxidation is only partially inhibited by chloride (Cl−), the presumed major physiological substrate for MPO. In fact, low concentrations of NO2− (2-10 μM) catalyze MPO-mediated oxidation of Cl−, indicated by increased chlorination of monochlorodimedon or 4-hydroxyphenylacetic acid, most likely via reduction of MPO
compound II. Peroxidase-catalyzed oxidation of NO2−, as indicated by phenolic nitration, was also observed in the presence of thiocyanate (SCN−), an alternative physiological substrate for mammalian peroxidases. Collectively, our results suggest that NO2−, at physiological or pathological levels, is a substrate for the mammalian peroxidases MPO and lactoperoxidase and that formation
of NO2· via peroxidase-catalyzed oxidation of NO2− may provide an additional pathway contributing to cytotoxicity or host defense associated with increased NO· production.
Ribonucleotide reductase is essential for DNA synthesis in cycling cells. It has been previously shown that the catalytically
competent tyrosyl free radical of its small R2 subunit (R2-Y⋅) is scavenged in tumor cells co-cultured with macrophages expressing a nitric oxide synthase II activity. We now demonstrate
a loss of R2-Y⋅ induced either by ⋅NO or peroxynitritein vitro. The ⋅NO effect is reversible and followed by an increase in ferric iron release from mouse protein R2. A similar increased iron
lability in radical-free, diferric metR2 protein suggests reciprocal stabilizing interactions between R2-Y⋅ and the diiron center in the mouse protein. Scavenging of R2-Y⋅ by peroxynitrite is irreversible and paralleled to an irreversible loss of R2 activity. Formation of nitrotyrosine and dihydroxyphenylalanine
was also detected in peroxynitrite-modified protein R2. In R2-overexpressing tumor cells co-cultured with activated murine
macrophages, scavenging of R2-Y⋅ following NO synthase II induction was fully reversible, even when endogenous production of peroxynitrite was induced by
triggering NADPH oxidase activity with a phorbol ester. Our results did not support the involvement of peroxynitrite in R2-Y⋅ scavenging by macrophage ⋅NO synthase II activity. They confirmed the preponderant physiological role of ⋅NO in the process.
Tyrosyl radicals have been detected during turnover of prostaglandin endoperoxide H synthase (PGHS), and they are speculated
to participate in cyclooxygenase catalysis. Spectroscopic approaches to elucidate the identity of the radicals have not been
definitive, so we have attempted to trap the radical(s) with nitric oxide (NO). NO quenched the EPR signal generated by reaction
of purified ram seminal vesicle PGHS with arachidonic acid, suggesting that NO coupled with a tyrosyl radical to form inter alianitrosocyclohexadienone. Subsequent formation of nitrotyrosine was detected by Western blotting of PGHS incubated with NO
and arachidonic acid or organic hydroperoxides using an antibody against nitrotyrosine. Both arachidonic acid and NO were
required to form nitrotyrosine, and tyrosine nitration was blocked by the PGHS inhibitor indomethacin. The presence of superoxide
dismutase had no effect on nitration, indicating that peroxynitrite was not the nitrating agent. To identify which tyrosines
were nitrated, PGHS was digested with trypsin, and the resulting peptides were separated by high pressure liquid chromatography
and monitored with a diode array detector. A single peptide was detected that exhibited a spectrum consistent with the presence
of nitrotyrosine. Consistent with Western blotting results, both NO and arachidonic acid were required to observe nitration
of this peptide, and its formation was blocked by the PGHS inhibitor indomethacin. Peptide sequencing indicated that the modified
residue was tyrosine 385, the source of the putative catalytically active tyrosyl radical.
The activity of glutamine synthetase (EC 6.3.1.2) from Escherichia coli is regulated by the cyclic adenylylation and deadenylylation of Tyr-397 in each of the enzyme's 12 identical subunits. The nitration of Tyr-397 or of the nearby Tyr-326 by peroxynitrite can convert the unadenylylated enzyme to a form exhibiting regulatory characteristics similar to the form obtained by adenylylation. The adenylylated conformation can also be elicited by the oxidation of surface-exposed methionine residues to methionine sulfoxide. However, the nitration of tyrosine residues and the oxidation of methionine residues are oppositely directed by the presence and absence of CO2. At physiological concentrations of CO2, pH 7.4, nitration occurs but oxidation of methionine residues is inhibited. Conversely, in the absence of CO2 methionine oxidation is stimulated and nitration of tyrosine is prevented. It was further established that adenylylation of Tyr-397 precludes its nitration by peroxynitrite. Furthermore, nitration of Tyr-326 together with either nitration or adenylylation of Tyr-397 leads to inactivation of the enzyme. These results demonstrate that CO2 can alter the course of peroxynitrite-dependent reactions and serve notice that (i) the reactions have physiological significance only if they are shown to occur at physiological concentrations of CO2 and physiological pH; and (ii) the peroxynitrite-dependent nitration of tyrosine residues or the oxidation of methionine residues of metabolically regulated proteins can seriously compromise their biological function.
P-selectin, a receptor for neutrophils and monocytes, is an adhesion molecule on the surface of activated platelets that resides in the alpha granule membrane of unstimulated platelets. To determine whether phosphorylation of P-selectin might accompany platelet activation, P-selectin in resting and thrombin-stimulated platelets labeled with o-[32P]phosphate was immunoprecipitated with the monoclonal antibody AC1.2 directed against P-selectin. SDS-gel electrophoresis of the immunoprecipitates indicated about 10-20-fold higher levels of 32P incorporated into P-selectin from thrombin-activated platelets than in resting platelets, although both sets of platelets contained equivalent amounts of P-selectin. The lower limits of the molar ratio of phosphate to P-selectin in activated platelets is about 0.52 +/- 0.08. Other platelet agonists, including the thrombin receptor peptide (SFLLR), epinephrine, ADP, and collagen, similarly stimulated phosphorylation of P-selectin. The kinetics of P-selectin phosphorylation following thrombin stimulation was rapid, with maximum phosphorylation observed at 15-30 s. Phosphoamino acid analysis of the phosphorylated P-selectin revealed the rapid synthesis of phosphoserine, phosphothreonine, and phosphotyrosine, but 80-90% of the phosphotyrosine and phosphothreonine disappeared within 5 min of platelet activation while the maximal level of phosphoserine remained stable. The rapid phosphorylation and selective dephosphorylation of specific amino acids in P-selectin following platelet activation may be important for P-selectin function and signal transduction within platelets.
Mitochondrial and cytosolic aconitases have been indicated as major targets of .NO- and O2-.-mediated toxicity in cells due to the oxidant-mediated disruption of the [4Fe-4S] prosthetic group. However, under circumstances in which both .NO and O2-. are generated, their almost diffusion-controlled combination reaction (k = 6.7 x 10(9) M-1 s-1), leading to the formation of peroxynitrite anion (ONOO-), can out-compete the direct reactions of .NO and O2-. with aconitase and even the enzymatic dismutation of O2-. by superoxide dismutase. In this work, we report that ONOO- reacts with isolated pig heart mitochondrial aconitase at 1.4 x 10(5) M-1 s-1, resulting in a significant loss of enzymatic activity. Aconitase activity was totally recovered after postincubation with thiols and ferrous iron, indicating that ONOO- reactions with the enzyme involve the perturbation of the labile Fe alpha to yield the inactive [3Fe-4S] cluster, which is also evident by spectral changes. On the other hand, anaerobic exposure of isolated aconitase to high concentrations of .NO (> 100 microM) led to a moderate inhibition of the enzyme, which could be fully overcome by .NO displacement under an argon-saturated atmosphere, in agreement with the formation of a reversible inhibitory complex between .NO and the active site of aconitase. Superoxide inactivated mitochondrial aconitase at (3.5 +/- 2) x 10(6) M-1 s-1, a reaction rate 3 orders of magnitude slower than its reaction rate with .NO. O2-. could represent the main mechanism of inactivation of the enzyme in systems in which it is formed without significant concomitant production of .NO. Our results imply that the mechanisms by which .NO and O2-. inactivate aconitase in cell systems may not be simple due to their direct reactions with the iron-sulfur cluster, but may rely on the formation of ONOO-.
Activated alveolar macrophages and epithelial type II cells release both nitric oxide and superoxide which react at near diffusion-limited rate (6.7 x 10(9) M-1s-1) to form peroxynitrite, a potent oxidant capable of damaging the alveolar epithelium and pulmonary surfactant. Peroxynitrite, but not nitric oxide or superoxide, readily nitrates phenolic rings including tyrosine. We quantified the presence of nitrotyrosine in the lungs of patients with the adult respiratory distress syndrome (ARDS) and in the lungs of rats exposed to hyperoxia (100% O2 for 60 h) using quantitative immunofluorescence. Fresh frozen or paraffin-embedded lung sections were incubated with a polyclonal antibody to nitrotyrosine, followed by goat anti-rabbit IgG coupled to rhodamine. Sections from patients with ARDS (n = 5), or from rats exposed to hyperoxia (n = 4), exhibited a twofold increase of specific binding over controls. This binding was blocked by the addition of an excess amount of nitrotyrosine and was absent when the nitrotyrosine antibody was replaced with nonimmune IgG. In additional experiments we demonstrated nitrotyrosine formation in rat lung sections incubated in vitro with peroxynitrite, but not nitric oxide or reactive oxygen species. These data suggest that toxic levels of peroxynitrite may be formed in the lungs of patients with acute lung injury.
The oxidation of lipoproteins is considered to play a key role in atherogenesis, and tyrosyl radicals have been implicated in the oxidation reaction. Tyrosyl radicals are generated in a system containing myeloperoxidase, H2O2, and tyrosine, but details of this enzyme-catalyzed reaction have not been explored. We have performed transient spectral and kinetic measurements to study the oxidation of tyrosine by the myeloperoxidase intermediates, compounds I and II, using both sequential mixing and single-mixing stopped-flow techniques. The one-electron reduction of compound I to compound II by tyrosine has a second order rate constant of (7.7 +/- 0.1) x 10(5) M-1 s-1. Compound II is then reduced by tyrosine to native enzyme with a second order rate constant of (1.57 +/- 0.06) x 10(4) M-1 s-1. Our study further revealed that, compared with horseradish peroxidase, thyroid peroxidase, and lactoperoxidase, myeloperoxidase is the most efficient catalyst of tyrosine oxidation at physiological pH. The second order rate constant for the myeloperoxidase compound I reaction with tyrosine is comparable with that of its compound I reaction with chloride: (4.7 +/- 0.1) x 10(6) M-1 s-1. Thus, although chloride is considered the major myeloperoxidase substrate, tyrosine is able to compete effectively for compound I. Steady state inhibition studies demonstrate that chloride binds very weakly to the tyrosine binding site of the enzyme. Coupling of tyrosyl radicals yields dityrosine, a highly fluorescent stable compound that had been identified as a possible marker for lipoprotein oxidation. We present spectral and kinetic data showing that dityrosine is further oxidized by both myeloperoxidase compounds I and II. The second order rate constants we determined for dityrosine oxidation are (1.12 +/- 0.01) x 10(5) M-1 s-1 for compound I and (7.5 +/- 0.3) x 10(2) M-1 s-1 for compound II. Therefore, caution must be exercised when using dityrosine as a quantitative index of lipoprotein oxidation, particularly in the presence of myeloperoxidase and H2O2.
Inflammatory processes in chronic rejection remain a serious clinical problem in organ transplantation. Activated cellular infiltrate produces high levels of both superoxide and nitric oxide. These reactive oxygen species interact to form peroxynitrite, a potent oxidant that can modify proteins to form 3-nitrotyrosine. We identified enhanced immunostaining for nitrotyrosine localized to tubular epithelium of chronically rejected human renal allografts. Western blot analysis of rejected tissue demonstrated that tyrosine nitration was restricted to a few specific polypeptides. Immunoprecipitation and amino acid sequencing techniques identified manganese superoxide dismutase, the major antioxidant enzyme in mitochondria, as one of the targets of tyrosine nitration. Total manganese superoxide dismutase protein was increased in rejected kidney, particularly in the tubular epithelium; however, enzymatic activity was significantly decreased. Exposure of recombinant human manganese superoxide dismutase to peroxynitrite resulted in a dose-dependent (IC50 = 10 microM) decrease in enzymatic activity and concomitant increase in tyrosine nitration. Collectively, these observations suggest a role for peroxynitrite during development and progression of chronic rejection in human renal allografts. In addition, inactivation of manganese superoxide dismutase by peroxynitrite may represent a general mechanism that progressively increases the production of peroxynitrite, leading to irreversible oxidative injury to mitochondria.
Oxidized low density lipoprotein (LDL) may be of central importance in triggering atherosclerosis. One potential pathway involves
the production of nitric oxide (NO) by vascular wall endothelial cells and macrophages. NO reacts with superoxide to form
peroxynitrite (ONOO−), a potent agent of LDL oxidation in vitro. ONOO− nitrates the aromatic ring of free tyrosine to produce 3-nitrotyrosine, a stable product. To explore the role of reactive
nitrogen species such as ONOO− in the pathogenesis of vascular disease, we developed a highly sensitive and specific method involving gas chromatography
and mass spectrometry to quantify 3-nitrotyrosine levels in proteins. In vitro studies demonstrated that 3-nitrotyrosine was a highly specific marker for LDL oxidized by ONOO−. LDL isolated from the plasma of healthy subjects had very low levels of 3-nitrotyrosine (9 ± 7 μmol/mol of tyrosine). In
striking contrast, LDL isolated from aortic atherosclerotic intima had 90-fold higher levels (840 ± 140 μmol/mol of tyrosine).
These observations strongly support the hypothesis that reactive nitrogen species such as ONOO− form in the human artery wall and provide direct evidence for a specific reaction pathway that promotes LDL oxidation in vivo. The detection of 3-nitrotyrosine in LDL isolated from vascular lesions raises the possibility that NO, by virtue of its
ability to form reactive nitrogen intermediates, may promote atherogenesis, counteracting the well-established anti-atherogenic
effects of NO.
The catalytic properties of glutathione reductase for its substrate, glutathione disulfide, were altered following a 60 s exposure to a 100-fold molar excess of peroxynitrite; the K(M) value was increased by approximately 2.5-fold and the V(max) value was decreased by approximately 1.7-fold. The kinetic alterations are thought to result from nitrotyrosine formation as the intrinsic Tyr fluorescence is diminished. The UV-visible spectrum of glutathione reductase exhibited absorbance at approximately 423 nm, characteristic of nitrotyrosine. In addition, the presence of nitrotyrosine has been detected by Western immunoblots with an anti-nitrotyrosine antibody. The peroxynitrite-induced inactivation is not observed in the presence of excess glutathione disulfide. However, excess NADPH offered no protection against peroxynitrite-induced inactivation. These observations suggest that the modification of approximately 1.8 Tyr per subunit, at or near the glutathione disulfide binding domain, probably results in the observed catalytic alterations. To test this hypothesis, the two tyrosines closest to the glutathione disulfide binding domain (Tyr114 and Tyr106), as indicated by the X-ray crystallographic data [Karplus and Schulz (1989) J. Biol. Chem., 210, 163-180], were each converted to nitrotyrosines by molecular modeling and the structure energy was minimized. These theoretical calculations indicate that the bond lengths between Tyr114-O and the Gly-N and Cys II-N of glutathione disulfide bound to glutathione reductase (Karplus and Schulz, 1989) increased by 3.0 and 4.3 A, respectively, upon nitration. In the case of Tyr106 the 0-Cys II-N distance also increases by approximately 1.6 A. The loss of these hydrogen bonding contacts is likely to result in the observed catalytic alterations upon reaction with peroxynitrite.
Flash photolysis of alkaline peroxynitrite solutions results in the formation of nitrogen monoxide and superoxide. From the rate of recombination it is concluded that the rate constant of the reaction of nitrogen monoxide with superoxide is (1.9 +/- 0.2) x 10(10) M-1 s-1. The pKa of hydrogen oxoperoxonitrate is dependent on the medium. With the stopped-flow technique a value of 6.5 is found at millimolar phosphate concentrations, while at 0.5 M phosphate the value is 7.5. The kinetics of decay do not follow first-order kinetics when the pH is larger than the pKa, combined with a total peroxynitrite and peroxynitrous acid concentration that exceeds 0.1 mM. An adduct between ONOO- and ONOOH is formed with a stability constant of (1.0 +/- 0.1) x 10(4) M. The kinetics of the decay of hydrogen oxoperoxonitrate are not very pressure-dependent: from stopped-flow experiments up to 152 MPa, an activation volume of 1.7 +/- 1.0 cm3 mol-1 was calculated. This small value is not compatible with homolysis of the O-O bond to yield free nitrogen dioxide and the hydroxyl radical. Pulse radiolysis of alkaline peroxynitrite solutions indicates that the hydroxyl radical reacts with ONOO- to form [(HO)ONOO].- with a rate constant of 5.8 x 10(9) M-1 s-1. This radical absorbs with a maximum at 420 nm (epsilon = 1.8 x 10(3) M-1 cm-1) and decays by second-order kinetics, k = 3.4 x 10(6) M-1 s-1. Improvements to the biomimetic synthesis of peroxynitrite with solid potassium superoxide and gaseous nitrogen monoxide result in higher peroxynitrite to nitrite yields than in most other syntheses.
We report that exposure of aconitase to moderate concentrations of peroxynitrite, 3-morpholinosydnonimine (SIN-1; a superoxide- and nitric oxide-liberating substance), or hydrogen peroxide, inhibits the enzyme and enhances susceptibility to proteolytic digestion by the isolated 20 S proteasome. Exposure to more severe levels of oxidative stress, from these same agents, causes further inhibition of the enzymatic activity of aconitase but actually decreases its proteolytic breakdown by proteasome. It should be noted that the superoxide and nitric oxide liberated by SIN-1 decomposition react to form a steady flux of peroxynitrite. S-Nitroso-N-acetylpenicillamine, a compound that liberates nitric oxide alone, causes only a small loss of aconitase activity (25% or less) and has no effect on the proteolytic susceptibility of the enzyme. Proteasome also seems to be the main protease in cell lysates that can degrade aconitase after it has been oxidatively modified by exposure to peroxynitrite, SIN-1, or hydrogen peroxide. Using cell lysates isolated from K562 cells treated for several days with an antisense oligodeoxynucleotide to the initiation codon region of the C2 subunit of proteasome (a treatment which diminishes proteasome activity by 50-60%), the enhanced degradation of moderately damaged aconitase was essentially abolished. Other model proteins as well as complex mixtures of proteins, such as cell lysates, also exhibit enhanced proteolytic susceptibility after moderate SIN-1 treatment. Therefore we conclude that peroxynitrite reacts readily with proteins and that mild modification by peroxynitrite results in selective recognition and degradation by proteasome.
The decrement in dopamine levels exceeds the loss of dopaminergic neurons in Parkinson's disease (PD) patients and experimental models of PD. This discrepancy is poorly understood and may represent an important event in the pathogenesis of PD. Herein, we report that the rate-limiting enzyme in dopamine synthesis, tyrosine hydroxylase (TH), is a selective target for nitration following exposure of PC12 cells to either peroxynitrite or 1-methyl-4-phenylpyridiniun ion (MPP+). Nitration of TH also occurs in mouse striatum after MPTP administration. Nitration of tyrosine residues in TH results in loss of enzymatic activity. In the mouse striatum, tyrosine nitration-mediated loss in TH activity parallels the decline in dopamine levels whereas the levels of TH protein remain unchanged for the first 6 hr post MPTP injection. Striatal TH was not nitrated in mice overexpressing copper/zinc superoxide dismutase after MPTP administration, supporting a critical role for superoxide in TH tyrosine nitration. These results indicate that tyrosine nitration-induced TH inactivation and consequently dopamine synthesis failure, represents an early and thus far unidentified biochemical event in MPTP neurotoxic process. The resemblance of the MPTP model with PD suggests that a similar phenomenon may occur in PD, influencing the severity of parkisonian symptoms.
The reaction of peroxynitrite (PN) with sarcomeric mitochondrial creatine kinase (Mib-CK; EC2.7.3.2) was observed at different stages of complexity (i) with purified Mi-CK, (ii) with enzyme bound on isolated mitoplasts, and
(iii) within intact respiring mitochondria. Creatine-stimulated respiration was abolished by PN concentrations likely to be
physiological and far before the respiratory chain itself was affected, thus demonstrating that Mi-CK is a prime target for
inactivation by PN in intact mitochondria. The inactivation by PN of Mi-CK was reversed by 22% with 2-mercaptoethanol. More
remarkable protective effects were noticed with the full set of CK substrates, e.g. 30 and 50% protection with MgATP plus creatine and MgADP plus phosphocreatine, respectively, but not with each substrate
alone. These data indicate an involvement of the active-site Cys-278 residue of Mi-CK in this process. Furthermore, changes
in endogenous tryptophan fluorescence intensity and spectral changes after reaction of Mi-CK with PN suggest additional modifications
of Trp and Tyr residues. PN-inactivated Mi-CK can no longer be efficiently converted into dimers by incubation with reagents
inducing a transition state analog complex at the active site. Thus, obviously, upon reaction of octameric Mi-CK with PN,
the octamer-dimer equilibrium of Mi-CK is also affected. The consequences for cellular energy homeostasis and calcium handling
are discussed.
Homogenates from rat spleen and lung could modify nitrotyrosine-containing BSA. With incubation, nitrotyrosine-containing BSA lost its epitope to a monoclonal antibody that selectively recognized nitrotyrosine-containing proteins. In the presence of protease inhibitors, the loss of the nitrotyrosine epitope occurred without protein degradation and hydrolysis. This activity was found in supernatant but not particulate fractions of spleen homogenates. The factor was heat labile, was sensitive to trypsin treatment, and was retained after passage through a membrane with a 10-kDa retention. The activity was time- and protein-concentration dependent. The activity increased about 2-fold in spleen extracts with endotoxin (bacterial lipopolysaccharide) treatment of animals, suggesting that the activity is inducible or regulatable. Other nitrotyrosine-containing proteins also served as substrates, while free nitrotyrosine and some endogenous nitrotyrosine-containing proteins in tissue extracts were poor substrates. Although the product and possible cofactors for this reaction have not yet been identified, this activity may be a "nitrotyrosine denitrase" that reverses protein nitration and, thus, decreases peroxynitrite toxicity. This activity was not observed in homogenates from rat liver or kidney, suggesting that there may also be some tissue specificity for the apparent denitrase activity.
NO2Tyr (3-Nitrotyrosine) is a modified amino acid that is formed by nitric oxide-derived species and has been implicated in the pathology of diverse human diseases. Nitration of active-site tyrosine residues is known to compromise protein structure and function. Although free NO2Tyr is produced in abundant concentrations under pathological conditions, its capacity to alter protein structure and function at the translational or posttranslational level is unknown. Here, we report that free NO2Tyr is transported into mammalian cells and selectively incorporated into the extreme carboxyl terminus of alpha-tubulin via a posttranslational mechanism catalyzed by the enzyme tubulin-tyrosine ligase. In contrast to the enzymatically regulated carboxyl-terminal tyrosination/detyrosination cycle of alpha-tubulin, incorporation of NO2Tyr shows apparent irreversibility. Nitrotyrosination of alpha-tubulin induces alterations in cell morphology, changes in microtubule organization, loss of epithelial-barrier function, and intracellular redistribution of the motor protein cytoplasmic dynein. These observations imply that posttranslational nitrotyrosination of alpha-tubulin invokes conformational changes, either directly or via allosteric interactions, in the surface-exposed carboxyl terminus of alpha-tubulin that compromises the function of this critical domain in regulating microtubule organization and binding of motor- and microtubule-associated proteins. Collectively, these observations illustrate a mechanism whereby free NO2Tyr can impact deleteriously on cell function under pathological conditions encompassing reactive nitrogen species production. The data also yield further insight into the role that the alpha-tubulin tyrosination/detyrosination cycle plays in microtubule function.
The accumulation of covalently modified proteins is an important hallmark of biological aging, but relatively few studies have addressed the detailed molecular-chemical changes and processes responsible for the modification of specific protein targets. Recently, Narayanan et al. [Narayanan, Jones, Xu and Yu (1996) Am. J. Physiol. 271, C1032-C1040] reported that the effects of aging on skeletal-muscle function are muscle-specific, with a significant age-dependent change in ATP-supported Ca2+-uptake activity for slow-twitch but not for fast-twitch muscle. Here we have characterized in detail the age-dependent functional and chemical modifications of the rat skeletal-muscle sarcoplasmic-reticulum (SR) Ca2+-ATPase isoforms SERCA1 and SERCA2a from fast-twitch and slow-twitch muscle respectively. We find a significant age-dependent loss in the Ca2+-ATPase activity (26% relative to Ca2+-ATPase content) and Ca2+-uptake rate specifically in SR isolated from predominantly slow-twitch, but not from fast-twitch, muscles. Western immunoblotting and amino acid analysis demonstrate that, selectively, the SERCA2a isoform progressively accumulates a significant amount of nitrotyrosine with age (approximately 3.5+/-0. 7 mol/mol of SR Ca2+-ATPase). Both Ca2+-ATPase isoforms suffer an age-dependent loss of reduced cysteine which is, however, functionally insignificant. In vitro, the incubation of fast- and slow-twitch muscle SR with peroxynitrite (ONOO-) (but not NO/O2) results in the selective nitration only of the SERCA2a, suggesting that ONOO- may be the source of the nitrating agent in vivo. A correlation of the SR Ca2+-ATPase activity and covalent protein modifications in vitro and in vivo suggests that tyrosine nitration may affect the Ca2+-ATPase activity. By means of partial and complete proteolytic digestion of purified SERCA2a with trypsin or Staphylococcus aureus V8 protease, followed by Western-blot, amino acid and HPLC-electrospray-MS (ESI-MS) analysis, we localized a large part of the age-dependent tyrosine nitration to the sequence Tyr294-Tyr295 in the M4-M8 transmembrane domain of the SERCA2a, close to sites essential for Ca2+ translocation.
Alzheimer's disease (AD) is a progressive amnestic disorder typified by the pathological misfolding and deposition of the microtubule-associated tau protein into neurofibrillary tangles (NFTs). While numerous post-translational modifications influence NFT formation, the molecular mechanisms responsible for tau aggregation remain enigmatic. Since nitrative and oxidative injury have previously been shown to play a mechanistic role in neurodegeneration, we examined whether these events influence tau aggregation. In this report, we characterize the effects of peroxynitrite (ONOO-)-mediated nitration and oxidation on tau polymerization in vitro. Treatment of tau with ONOO- results in 3-nitrotyrosine (3-NT) immunoreactivity and the formation of heat-stable, SDS-insoluble oligomers. Using ESI-MS and HPLC with fluorescent detection, we show that these higher-order aggregates contain 3,3'-dityrosine (3,3'-DT). Tyrosine (Tyr) residues are critical for ONOO(-)-mediated oligomerization, as tau proteins lacking all Tyr residues fail to generate oligomers upon ONOO- treatment. Further, tau nitration targets residues Y18, Y29, and to a lesser degree Y197 and Y394, and nitration at these sites inhibits in vitro polymerization. The inhibitory effect of nitration on tau polymerization is specific for the 3-NT modification, as pseudophosphorylation at these same Tyr residues does not inhibit tau assembly. Our results suggest that the nitrative and oxidative roles of ONOO- differentially affect tau polymerization and that ONOO(-)-mediated cross-linking could facilitate tau aggregation in AD.
The addition of peroxynitrite to purified cytochrome P450 2B1 resulted in a concentration-dependent loss of the NADPH- and reductase-supported or tert-butylhydroperoxide-supported 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of P450 2B1 with IC50 values of 39 and 210 μM, respectively. After incubation of P450 2B1 with 300 μM peroxynitrite, the heme moiety was not altered, but the apoprotein was modified as shown by HPLC and spectral analysis. Western blot analysis of peroxynitrite-treated P450 2B1 demonstrated the presence of an extensive immunoreactivite band after incubating with anti-nitrotyrosine antibody. However, the immunostaining was completely abolished after coincubation of the anti-nitrotyrosine antibody with 10 mM nitrotyrosine. These results indicated that one or more of the tyrosine residues in P450 2B1 were modified to nitrotyrosines. The decrease in the enzymatic activity correlated with the increase in the extent of tyrosine nitration. Further demonstration of tyrosine nitration was confirmed by GC/MS analysis by using 13C-labeled tyrosine and nitrotyrosine as internal standards; approximately 0.97 mol of nitrotyrosine per mole of P450 2B1 was found after treatment with peroxynitrite. The peroxynitrite-treated P450 2B1 was digested with Lys C, and the resulting peptides were separated by Tricine-sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE). The amino acid sequence of the major nitrotyrosine-containing peptide corresponded to a peptide containing amino acid residues 160−225 of P450 2B1, which contains two tyrosine residues. Thus, incubation of P450 2B1 with peroxynitrite resulted in the nitration of tyrosines at either residue 190 or 203 or at both residues of P450 2B1 concomitant with a loss of 2B1-dependent activity.
Peroxynitrite is a strong oxidant involved in cell injury. In tissues, most of peroxynitrite reacts preferentially with CO2 or hemoproteins, and these reactions affect its fate and toxicity. CO2 promotes tyrosine nitration but reduces the lifetime of peroxynitrite, preventing, at least in part, membrane crossing. The role of hemoproteins is not easily predictable, because the heme intercepts peroxynitrite, but its oxidation to ferryl species and tyrosyl radical(s) may catalyze tyrosine nitration. The modifications induced by peroxynitrite/CO2 on oxyhemoglobin were determined by mass spectrometry, and we found that αTyr42, βTyr130, and, to a lesser extent, αTyr24 were nitrated. The suggested nitration mechanism is tyrosyl radical formation by long-range electron transfer to ferrylhemoglobin followed by a reaction with •NO2. Dityrosine (α24−α42) and disulfides (β93−β93 and α104−α104) were also detected, but these cross-linkings were largely due to modifications occurring under the denaturing conditions employed for mass spectrometry. Moreover, immunoelectrophoretic techniques showed that the 3-nitrotyrosine content of oxyhemoglobin sharply increased only in molar excess of peroxynitrite, thus suggesting that this hemoprotein is not a catalyst of nitration. The noncatalytic role may be due to the formation of the nitrating species •NO2 mainly in molar excess of peroxynitrite. In agreement with this hypothesis, oxyhemoglobin strongly inhibited tyrosine nitration of a target dipeptide (Ala−Tyr) and of membrane proteins from ghosts resealed with oxyhemoglobin. Erythrocytes were poor inhibitors of Ala−Tyr nitration on account of the membrane barrier. However, at the physiologic hematocrit, Ala−Tyr nitration was reduced by 65%. This “sink” function was facilitated by the huge amount of band 3 anion exchanger on the cell membrane. We conclude that in blood oxyhemoglobin is a peroxynitrite scavenger of physiologic relevance.
Cytochrome c3+-catalyzed peroxidation of phosphatidylcholine liposomes by hydrogen peroxide (H2O2) was indicated by the production of thiobarbituric acid reactive substances, oxygen consumption, and emission of spontaneous chemiluminescence. The iron chelator diethylenetriaminepentaacetic acid (DTPA) only partially inhibited peroxidation when H2O2 concentrations were 200 μm or greater. In contrast, iron compounds such as ferric chloride, potassium ferricyanide, and hemin induced H2O2-dependent lipid peroxidation which was totally inhibitable by DTPA. Cyanide and urate, which react at or near the cytochrome—heme, completely prevented lipid peroxidation, while hydroxyl radical scavengers and superoxide dismutase had very little or no inhibitory effect. Changes in liposome surface charge did not influence cytochrome c3+ plus H2O2-dependent peroxidation, but a net negative charge was critical in favoring cytochrome c3+-dependent, H2O2-independent lipid auto-oxidative processes. These results show that reaction of cytochrome c with H2O2 promotes membrane oxidation by more than one chemical mechanism, including formation of high oxidation states of iron at the cytochrome—heme and also by heme iron release at higher H2O2 concentrations. Cytochrome c3+ could react with mitochondrial H2O2 to yield “site-specific” mitochondrial membrane lipid peroxidation during tissue oxidant stress.
Human recombinant MnSOD and CuZnSOD were both inactivated when exposed to simultaneous fluxes of superoxide (JO(2)(*-)) and nitric oxide (J*NO). The inactivation was also observed with varying J*NO/JO(2)(*-) ratios. Protein-derived radicals were detected in both CuZn and MnSOD by immuno-spin trapping. The formation of protein radicals was followed by tyrosine nitration in the case of MnSOD. When MnSOD was exposed to J*NO and JO(2)(*-) in the presence of uric acid, a scavenger of peroxynitrite-derived free radicals, nitration was decreased but inactivation was not prevented. On the other hand, glutathione, known to react with both peroxynitrite and nitrogen dioxide, totally protected MnSOD from inactivation and nitration on addition of authentic peroxynitrite but, notably, it was only partially inhibitory in the presence of the more biologically relevant J*NO and JO(2)(*-). The data are consistent with the direct reaction of peroxynitrite with the Mn center and a metal-catalyzed nitration of Tyr-34 in MnSOD. In this context, we propose that inactivation is also occurring through a *NO-dependent nitration mechanism. Our results help to rationalize MnSOD tyrosine nitration observed in inflammatory conditions in vivo in the presence of low molecular weight scavengers such as glutathione that otherwise would completely consume nitrogen dioxide and prevent nitration reactions.
Peroxynitrite formation by rat alveolar macrophages activated with phorbol 12-myristate 13-acetate was assayed by the Cu,Zn superoxide dismutase-catalyzed nitration of 4-hydroxyphenylacetate. The inhibitor of nitric oxide synthesis N-methyl-L-arginine prevented the Cu,Zn superoxide dismutase-catalyzed nitration of 4-hydroxyphenylacetate by stimulated macrophages, while Cu-depleted Zn superoxide dismutase did not catalyze the formation of 3-nitro-4-hydroxyphenylacetate either in vitro or in the presence of activated macrophages. The rate of phenolic nitration by activated macrophages was 9 +/- 2 pmol x 10(6) cells-1 x min-1 (mean +/- STD). Only 8% of synthetic peroxynitrite was trapped by superoxide dismutase, which suggested that the rate of peroxynitrite formation may have been as high as 0.11 nmol x 10(6) cells-1 x min-1. This upper estimate was consistent with N-methyl-L-arginine increasing the amount of superoxide detected with cytochrome c by 0.12 nmol x 10(6) cells-1 x min-1. The rate of nitrite and nitrate accumulation was 0.10 +/- 0.001 nmol x 10(6) cells-1 x min-1, suggesting that the majority of nitric oxide produced by activated macrophages may have been converted to peroxynitrite. The formation of a relatively long lived, strong oxidant from the reaction of nitric oxide and superoxide in activated macrophages may contribute to inflammatory cell-mediated tissue injury.
Superoxide dismutase and Fe3+EDTA catalyzed the nitration by peroxynitrite (ONOO-) of a wide range of phenolics including tyrosine in proteins. Nitration was not mediated by a free radical mechanism because hydroxyl radical scavengers did not reduce either superoxide dismutase or Fe3+EDTA-catalyzed nitration and nitrogen dioxide was not a significant product from either catalyst. Rather, metal ions appear to catalyze the heterolytic cleavage of peroxynitrite to form a nitronium-like species (NO2+). The calculated energy for separating peroxynitrous acid into hydroxide ion and nitronium ion is 13 kcal.mol-1 at pH 7.0. Fe3+EDTA catalyzed nitration with an activation energy of 12 kcal.mol-1 at a rate of 5700 M-1.s-1 at 37 degrees C and pH 7.5. The reaction rate of peroxynitrite with bovine Cu,Zn superoxide dismutase was 10(5) M-1.s-1 at low superoxide dismutase concentrations, but the rate of nitration became independent of superoxide dismutase concentration above 10 microM with only 9% of added peroxynitrite yielding nitrophenol. We propose that peroxynitrite anion is more stable in the cis conformation, whereas only a higher energy species in the trans conformation can fit in the active site of Cu,Zn superoxide dismutase. At high superoxide dismutase concentrations, phenolic nitration may be limited by the rate of isomerization from the cis to trans conformations of peroxynitrite as well as by competing pathways for peroxynitrite decomposition. In contrast, Fe3+EDTA appears to react directly with the cis anion, resulting in greater nitration yields.
The crystal structure of bovine Cu,Zn superoxide dismutase modified with peroxynitrite (ONOO-) was determined by X-ray diffraction, utilizing the existing three-dimensional model of the native structure deposited in the Brookhaven Protein Data Bank (J. A. Tainer et al., J. Mol. Biol. 160, 181-217, 1982). The native structure and the modified derivative were refined to R factors of 19.0 and 18.7% respectively using diffraction data from 6.0 to 2.5 A. The major result after reaction with peroxynitrite was the appearance of electron density 1.45 A from a single epsilon carbon of Tyr-108, the only tyrosine residue in the sequence. Tyr-108 is a solvent-exposed residue 18 A from the copper atom in the active site. The electron density was consistent with nitration of Tyr-108 at one of the epsilon carbons to form 3-nitrotyrosine. We propose that the nitration occurs in solution by transfer of a nitronium-like species from the active site on one superoxide dismutase dimer to the Tyr-108 of a second dimer.
3-Nitrotyrosine (NTYR) in tissue or blood proteins was evaluated as a possible exposure marker for exogenous and endogenous nitrosating or nitrating agents. A sensitive and selective method for analysing NTYR by gas chromatography with a thermal energy analyser (GC-TEA) was developed. Using this method, a number of kinetic studies were carried out. It was found that free and protein-bound tyrosine residues easily react with nitrating/nitrosating agents to yield NTYR. NTYR formation in vivo showed a dose-dependent increase in NTYR in both plasma proteins and haemoglobin obtained from rats 24 hr after ip injection of various doses (0.5-2.5 mumol/rat) of tetranitromethane. Major urinary metabolites of NTYR, given orally to rats, were isolated and identified as 3-nitro-4-hydroxyphenylacetic acid (NHPA) and 3-nitro-4-hydroxyphenyllactic acid (NHPL). About 44% and 5% of the oral dose of NTYR (100 micrograms/rat) was excreted as NHPA and NHPL, respectively. Eleven 24-hr human urine samples were analysed for NHPA by GC-TEA after ethyl acetate extraction and HPLC purification: quantities ranging from 0 to 7.9 micrograms/24 hr, mean +/- SD 2.8 +/- 2.3 (n = 11) were detected (detection limit 0.2 micrograms/litre). NTYR in proteins or its metabolites in urine can be readily analysed by GC-TEA as a new/additional marker for endogenous nitrosation and nitration.
In recent years it has become clear that various free radicals and related oxidants can cause serious damage to intracellular enzymes and other proteins. Several investigators have shown that in extreme cases this can result in an accumulation of oxidatively damaged proteins as useless cellular debris. In other instances, proteins may undergo scission reactions with certain radicals/oxidants, resulting in the direct formation of potentially toxic peptide fragments. Data has also been gathered (recently) demonstrating that various intracellular proteolytic enzymes or systems can recognize, and preferentially degrade, oxidatively damaged proteins (to amino acids). In this hypothesis paper I present evidence to suggest that proteolytic systems (of proteinases, proteases, and peptidases) may function to prevent the formation or accumulation of oxidatively damaged protein aggregates. Proteolytic systems can also preferentially degrade peptide fragments and may thus prevent a wide variety of potentially toxic consequences. I propose that many proteolytic enzymes may be important components of overall antioxidant defenses because they can act to ameliorate the consequences of oxidative damage. A modified terminology is suggested in which the primary antioxidants are such agents as vitamin E, beta-carotene, and uric acid and such enzymes as superoxide dismutase, glutathione peroxidase, and DT-diaphorase. In this classification scheme, proteolytic systems, DNA repair systems, and certain lipolytic enzymes would be considered as secondary antioxidant defenses. As secondary antioxidant defenses, proteolytic systems may be particularly important in times of high oxidative stress, during periods of (primary) antioxidant insufficiency, or with advancing age.
A series of radical transitions, Br2-.----Met(S therefore Br)----Trp(indolyl)----Tyr (phenoxyl), has been demonstrated by pulse radiolysis of N2O-saturated aqueous solutions containing Br-, Met-Gly and Trp-(Gly)2-Tyr at pH 6.7. The intramolecular Met(S therefore Br)----Trp(indolyl) transition in the dipeptide Met-Trp is shown to proceed via the Trp+. radical cation, with a rate constant of k approximately 10(7)s-1, consistent with an electron transfer. Br2-.-attack upon ribonuclease A (RNase) leads to a fast Met(S therefore Br)----Tyr(phenoxyl) process, k = (4.0 +/- 1.0) X 10(5)s-1, probably involving the solvent-exposed Met-29 and the adjacent Tyr-25. Phenoxyl dimerization in the RNase system produces the characteristic o,o'-biphenol fluorescence, but a competing interaction of the Tyr-25(phenoxyl) with the 26-84 disulphide group also appears possible.
Cytochrome c3+ has been extensively used for the detection of superoxide produced in biological systems due to its fast superoxide-mediated reduction to cytochrome c2+. However, another biomolecule which is sometimes cogenerated with superoxide, nitric oxide, reacts with superoxide at almost diffusion-controlled rates (6.7 x 10(9) M-1 s-1), leading to the production of a highly oxidizing species, peroxynitrite anion (ONOO-). In this work we report that peroxynitrite readily oxidizes cytochrome c2+ to cytochrome c3+ in an ascorbate-reversible manner. The reaction between peroxynitrite and cytochrome c2+ occurs with a second-order rate constant of 2.3 x 10(5) M-1 s-1. The pH dependence of the apparent second-order rate constants as well as the effect of different scavengers indicated that peroxynitrous acid (ONOOH) in the ground state was the actual species responsible of cytochrome c2+ oxidation. The activation enthalpy, free energy, and entropy were +10.8 kcal mol-1, +11.8 kcal mol-1, and -3.15 cal mol-1 K-1, respectively, in agreement with the proposed reaction mechanism. Additionally, our results imply that when quantitating superoxide by the cytochrome c3+ reduction method, the existence of a simultaneous generation of nitric oxide and peroxynitrite may lead to an underestimation of the rates of superoxide production.
Phagocytes generate H2O2 for use by a secreted heme enzyme, myeloperoxidase, to kill invading bacteria, viruses, and fungi. We have explored the possibility that myeloperoxidase might also convert L-tyrosine to a radical catalyst that cross-links proteins. Protein-bound tyrosyl residues exposed to myeloperoxidase, H2O2, and L-tyrosine were oxidized to o,o'-dityrosine, a stable product of the tyrosyl radical. The cross-linking reaction required L-tyrosine but was independent of halide and free transition metal ions; the heme poisons azide and aminotriazole were inhibitory. Activated neutrophils likewise converted polypeptide tyrosines to dityrosine. The pathway for oxidation of peptide tyrosyl residues was dependent upon L-tyrosine and was inhibited by heme poisons and catalase. Dityrosine synthesis was little affected by plasma concentrations of Cl- and amino acids, suggesting that the reaction pathway might be physiologically relevant. The requirement for free L-tyrosine and H2O2 for dityrosine formation and the inhibition by heme poisons support the hypothesis that myeloperoxidase catalyzes the cross-linking of proteins by a peroxidative mechanism involving tyrosyl radical. In striking contrast to the pathways generally used to study protein oxidation in vitro, the reaction does not require free metal ions. We speculate that protein dityrosine cross-linking by myeloperoxidase may play a role in bacterial killing or injuring normal tissue. The intense fluorescence and stability of biphenolic compounds may allow dityrosine to act as a marker for proteins oxidatively damaged by myeloperoxidase in phagocyte-rich inflammatory lesions.
To determine if nitration of tyrosine residues by peroxynitrite (PN), which can be generated endogenously, can disrupt the phosphorylation of tyrosine residues in proteins involved in cell signaling networks, we studied the effect of PN-promoted nitration of tyrosine residues in a pentadecameric peptide, cdc2(6-20)NH2, on the ability of the peptide to be phosphorylated. cdc2(6-20)NH2 corresponds to the tyrosine phosphorylation site of p34cdc2 kinase, which is phosphorylated by lck kinase (lymphocyte-specific tyrosine kinase, p56lck). PN nitrates both Tyr-15 and Tyr-19 of the peptide in phosphate buffer (pH 7.5) at 37 degrees C. Nitration of Tyr-15. which is the phosphorylated amino acid residue, inhibits completely the phosphorylation of the peptide. The nitration reaction is enhanced by either Fe(III)EDTA or Cu(II)-Zn(II)-superoxide dismutase (Cu,Zn-SOD). The kinetic data are consistent with the view that reactions of Fe(111)EDTA or Cu,Zn-SOD with the cis form of PN yield complexes in which PN decomposes more slowly to form N02+, the nitrating agent. Thus, the nitration efficiency of PN is enhanced. These results are discussed from the point of view that PN-promoted nitration will result in permanent impairment of cyclic cascades that control signal transduction processes and regulate cell cycles.
The ability of protein tyrosine kinases to phosphorylate a synthetic peptide was inhibited 51% by peroxynitrite-mediated nitration of tyrosine. Exposure of endothelial cells to peroxynitrite decreased the intensity of tyrosine phosphorylated proteins and increased the intensity of nitrotyrosine-containing proteins. Peroxynitrite-modified BSA was degraded by human red blood cell lysates. However, human plasma in a concentration-, time-, and temperature-dependent manner, removed the protein nitrotyrosine epitope. These results suggest that tyrosine nitration interferes with phosphorylation and targets proteins for degradation. Specific enzymatic process(es) for removing nitrotyrosine may be present in vivo.
This chapter discusses the antibodies that recognize nitrotyrosine. Peroxynitrite is a potent oxidant that reacts with a variety of biological molecules, oxidizing thiols, initiating lipid peroxidation, inactivating ion channels, and damaging DNA. Tyrosine nitration can inactivate enzymes and receptors that depend on tyrosine residues for their activity. Nitration prevents phosphorylation of tyrosine residues important for signal transduction. Nitration of phenolics by peroxynitrite occurs spontaneously however is also catalyzed by low molecular mass transition metals and by superoxide dismutase and other metalloproteins. Nitrotyrosine in proteins occurs spontaneously when peroxynitrite is added to purified solutions of protein. The existence of nitrotyrosine is important indirect evidence that peroxynitrite is involved in pathophysiological processes. Nitration of tyrosine residues in purified nonheme proteins is relatively easy to detect by visible spectroscopy owing to the characteristic yellow color. Nitrotyrosine can be identified by gas chromatography, mass spectroscopy, and high-performance liquid chromatography (HPLC). Peroxynitrite is prepared by quenching the reaction of acidified nitrite and hydrogen peroxide with an excess of sodium hydroxide.
The proteasome is an essential component of the ATP-dependent proteolytic pathway in eukaryotic cells and is responsible for the degradation of most cellular proteins. The 20S (700-kDa) proteasome contains multiple peptidase activities that function through a new type of proteolytic mechanism involving a threonine active site. The 26S (2000-kDa) complex, which degrades ubiquitinated proteins, contains in addition to the 20S proteasome a 19S regulatory complex composed of multiple ATPases and components necessary for binding protein substrates. The proteasome has been highly conserved during eukaryotic evolution, and simpler forms are even found in archaebacteria and eubacteria. Major advances have been achieved recently in our knowledge about the molecular organization of the 20S and 19S particles, their subunits, the proteasome's role in MHC-class 1 antigen presentation, and regulators of its activities. This article focuses on recent progress concerning the biochemical mechanisms and intracellular functions of the 20S and 26S proteasomes.
Previous studies have shown that exposure of human surfactant protein A (SP-A) to nitrating agents [peroxynitrite (ONOO-); tetranitromethane (TNM; pH 8)] leads to nitrotyrosine formation. However, specific sites of nitration have not been identified. Herein, human SP-A, dissolved in Hepes buffer, was incubated with two boluses each of 0.5 mM ONOO- (pH 7.4) or 0.5 mM TNM (pH 8.0) for 15 min. After 30 min, SP-A samples were reduced, alkylated, and trypsin digested. The nitrated peptides and sites of amino acid nitration on the protein were identified by capillary high-performance liquid chromatography-coupled electrospray ionization tandem mass spectrometry (LC-ESMS/MS). The major nitrated peptide on both TNM- and (ONOO-)-exposed SP-A was the tryptic fragment Tyr161-Arg179 (YNTYAYVGLTEGPSPGDFR), located in the SP-A carbohydrate recognition domain. Sequencing of this nitrated peptide by LC-ESMS/MS demonstrated that the nitration was equally distributed on Tyr164 and Tyr166. A second lesser nitrated peptide corresponding to tryptic fragment Asn217-Arg222 (NCLYSR) was also found on TNM- and (ONOO-)-modified SP-A. No other nitrated amino acid was detected. Nitrated SP-A exhibited decreased ability to aggregate surfactant lipids in the presence of Ca2+. These data demonstrate that nitration of a specific tyrosine decreased an important protein function.
Vascular tone critically depends on the endothelial release of nitric oxide and prostacyclin. Superoxide anions counteract these relaxations by trapping nitric oxide under formation of peroxynitrite. As we have recently reported, peroxynitrite is able to inhibit prostacyclin formation in aortic microsomes (Zou et al., 1996). Here we show that peroxynitrite also blocks purified prostacyclin synthase with an IC50 value of about 50 nM and with a similar sensitivity also inhibits the enzyme activity in the EaHy 926 endothelial cell line. Thromboxane synthase, having the same heme-thiolate (P450) structure and a closely-related mechanism was unaffected by peroxynitrite. Anti-nitrotyrosine antibodies reacted positive by a Western blot after treatment of the purified enzyme with 1 microM peroxynitrite. Tetranitromethane also inhibited the enzyme activity which, like the inhibition by peroxynitrite, could be partially prevented in the presence of the substrate analog U46619. The simultaneous generation of superoxide and nitric oxide proved to be as efficient as a bolus of peroxynitrite which supports a possible inactivation of prostacyclin synthase under in vivo conditions. This substantiates an often suggested crucial role of superoxide in the pathophysiology of the cardiovascular system.
Superoxide dismutase (SOD) catalyzes the nitration of specific tyrosine residues in proteins by peroxynitrite (ONOO-), which may be the damaging gain-of-function resulting from mutations to SOD associated with familial amyotrophic lateral sclerosis (ALS). We found that disassembled neurofilament-L (light subunit) was more susceptible to tyrosine nitration catalyzed by SOD in vitro. Neurofilament-L was selectively nitrated compared with the majority of other proteins present in brain homogenates. Assembled neurofilament-L was more resistant to nitration, suggesting that the susceptible tyrosine residues were protected by intersubunit contacts in assembled neurofilaments. Electrospray mass spectrometry of trypsin-digested neurofilament-L showed that tyrosine 17 in the head region and tyrosines 138, 177, and 265 in alpha-helical coil regions of the rod domain of neurofilament-L were particularly susceptible to SOD-catalyzed nitration. Nitrated neurofilament-L inhibited the assembly of unmodified neurofilament subunits, suggesting that the affected tyrosines are located in regions important for intersubunit contacts. Neurofilaments are major structural proteins expressed in motor neurons and known to be important for their survival in vivo. We suggest that SOD-catalyzed nitration of neurofilament-L may have a significant role in the pathogenesis of ALS.
Lewy bodies (LBs) are hallmark lesions of degenerating neurons in the brains of patients with Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Recently, a point mutation in the gene encoding the presynaptic alpha-synuclein protein was identified in some autosomal-dominantly inherited familial PD pedigrees, and light microscopic studies demonstrated alpha-synuclein immunoreactivity in LBs of sporadic PD and DLB. To characterize alpha-synuclein in LBs, we raised monoclonal antibodies (MAbs) to LBs purified from DLB brains and obtained a MAb specific for alpha-synuclein that intensely labeled LBs. Light and electron microscopic immunocytochemical studies performed with this MAb as well as other antibodies to alpha-and beta-synuclein showed that alpha-synuclein, but not beta-synuclein, is a component of LBs in sporadic PD and DLB. Western blot analyses of highly purified LBs from DLB brains showed that full-length as well as partially truncated and insoluble aggregates of alpha-synuclein are deposited in LBs. Thus, these data strongly implicate alpha-synuclein in the formation of LBs and the selective degeneration of neurons in sporadic PD and DLB.
The technique of pulse radiolysis has been used to investigate the possibility of intramolecular charge transfer in the dipeptide histidyltyrosine, following one-electron oxidation of one of its amino acid residues. The radical anion, Br2.- was found to react with the dipeptide at pH 6.0 with a bimolecular rate constant of 2.3+/-0.2 x 10(7) dm3 mol(-1)s(-1) suggesting that it reacts very selectively with the histidine moiety. Spectral observations at, or close to the end of this reaction show only the presence of a tyrosinyl free radical (TyrO.), however, indicating that fast (>10(6) s(-1) intramolecular charge transfer has taken place between histidine radicals (His+.) and tyrosine (TyrOH). This finding was supported by the direct observation of the rate of formation of TyrO. in experiments with the free amino acids, histidine and tyrosine, under conditions where Br2.- reacted selectively with histidine. The bimolecular rate constant for the reaction between His+. and TyrOH was found to be 2.4+/-0.5 x 10(6) dm3 mol(-1)s(-1). Taken together, the results of the study indicate that His+. is a relatively strong oxidising agent where (E (His+./His) > 770 mV at pH 6.0.
Spin-trapping with 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) was used to demonstrate that 3-nitrotyrosine (nitrotyrosine) promotes the formation of substantial amounts of reactive oxygen species (O2.- and *OH), when incubated with NAD(H)-cytochrome c reductase and a corresponding electron donor. Spin adduct formation is strongly inhibited by the presence of superoxide dismutase (SOD); spin adduct formation requires aerobic conditions. Nitration of leucine enkephalin, a tyrosine-containing pentapeptide, results in a similar generation of O2*- and *OH species. Both nitrotyrosine and nitrated leucine enkephalin stimulate acetylated ferricytochrome c reduction in the presence of NAD(H)-cytochrome c reductase with typical Michaelis-Menten kinetics and Km's of 104 +/- 14 and 0.78 +/- 0.11 microM, respectively. No stimulation of acetylated ferricytochrome c reduction is observed in the presence of SOD. Catalase and the metal chelators DTPA and deferoxamine mesylate do not influence observed stimulation of acetylated ferricytochrome c reduction by nitrotyrosine. Nitration of two tyrosines (of four) within the sequence of the 6.5-kDa globular protein bovine pancreas trypsin inhibitor (BPTI) fails to stimulate O2*- generation implying steric restrictions for BPTI-reductase interactions. However, nitrated BPTI subjected to trypsin digestion stimulated reduction of acetylated ferricytochrome c. These results suggest that, as with other nitroaromatic compounds, nitrotyrosine may be enzymatically reduced to the corresponding nitro anion radical (ArNO2*-) which is then oxidized by molecular oxygen to yield O2*- and regenerate ArNO2. Thus, once formed in vivo, nitrotyrosine may act to promote oxidative stress by means of repetitive redox cycling.
Peroxynitrite has recently been implicated in the inactivation of many enzymes. However, little has been reported on the structural
basis of the inactivation reaction. This study proposes that nitration of a specific tyrosine residue is responsible for inactivation
of recombinant human mitochondrial manganese-superoxide dismutase (Mn-SOD) by peroxynitrite. Mass spectroscopic analysis of
the peroxynitrite-inactivated Mn-SOD showed an increased molecular mass because of a single nitro group substituted onto a
tyrosine residue. Single peptides that had different elution positions between samples from the native and peroxynitrite-inactivated
Mn-SOD on reverse-phase high performance liquid chromatography were isolated after successive digestion of the samples by
staphylococcal serine protease and lysylendopeptidase and subjected to amino acid sequence and molecular mass analyses. We
found that tyrosine 34 of the enzyme was exclusively nitrated to 3-nitrotyrosine by peroxynitrite. This residue is located
near manganese and in a substrate O⨪2 gateway in Mn-SOD.
Analytical and immunological methodologies and occasionally both methodologies have been applied to detect and quantify 3-nitrotyrosine in almost every major organ system. In certain diseases increased levels of 3-nitrotyrosine have been correlated with elevated levels of other indices of oxidative stress. Numerous reports have established that nitration is a biological process derived from the biochemical interaction of nitric oxide or nitric oxide-derived secondary products with reactive oxygen species. This article addresses critical issues regarding this biological process, namely the biochemical pathways for nitration of tyrosine residues in vivo, potential protein targets, and pathophysiological consequences of protein tyrosine nitration.
Oxidatively modified ferritin is selectively recognized and degraded by the 20S proteasome. Concentrations of hydrogen peroxide (H2O2) higher than 10 micromol/mg of protein are able to prevent proteolytic degradation. Exposure of the protease to high amounts of oxidants (H2O2, peroxynitrite and hypochlorite) inhibits the enzymic activity of the 20S proteasome towards the fluorogenic peptide succinyl-leucine-leucine-valine-tyrosine-methylcoumarylamide (Suc-LLVY-MCA), as well as the proteolytic degradation of normal and oxidant-treated ferritin. Fifty per cent inhibition of the degradation of the protein substrates was achieved using 40 micromol of H2O2/mg of proteasome. No change in the composition of the enzyme was revealed by electrophoretic analysis up to concentrations of 120 micromol of H2O2/mg of proteasome. In further experiments, it was found that the 26S proteasome, the ATP- and ubiquitin-dependent form of the proteasomal system, is much more susceptible to oxidative stress. Whereas degradation of the fluorogenic peptide, Suc-LLVY-MCA, by the 20S proteasome was inhibited by 50% with 12 micromol of H2O2/mg, 3 micromol of H2O2/mg was enough to inhibit ATP-stimulated degradation by the 26S proteasome by 50%. This loss in activity could be followed by the loss of band intensity in the non-denaturing gel. Therefore we concluded that the 20S proteasome was more resistant to oxidative stress than the ATP- and ubiquitin-dependent 26S proteasome. Furthermore, we investigated the activity of both proteases in K562 cells after H2O2 treatment. Lysates from K562 cells are able to degrade oxidized ferritin at a higher rate than non-oxidized ferritin, in an ATP-independent manner. This effect could be followed even after treatment of the cells with H2O2 up to a concentration of 2mM. The lactacystin-sensitive ATP-stimulated degradation of the fluorogenic peptide Suc-LLVY-MCA declined, after treatment of the cells with 1mM H2O2, to the same level as that obtained without ATP stimulation. Therefore, we conclude that the regulation of the 20S proteasome by various regulators takes place during oxidative stress. This provides further evidence for the role of the 20S proteasome in the secondary antioxidative defences of mammalian cells.
Peroxynitrite (ONOO-) is a potent oxidizing and nitrating agent produced by the reaction of nitric oxide with superoxide. It readily nitrates phenolic compounds such as tyrosine residues in proteins, and it has been demonstrated that nitration of tyrosine residues in proteins inhibits their phosphorylation. During immune responses, tyrosine phosphorylation of key substrates by protein tyrosine kinases is the earliest of the intracellular signaling pathways following activation through the TCR complex. This work was aimed to evaluate the effects of ONOO- on lymphocyte tyrosine phosphorylation, proliferation, and survival. Additionally, we studied the generation of nitrating species in vivo and in vitro during immune activation. Our results demonstrate that ONOO-, through nitration of tyrosine residues, is able to inhibit activation-induced protein tyrosine phosphorylation in purified lymphocytes and prime them to undergo apoptotic cell death after PHA- or CD3-mediated activation but not upon phorbol ester-mediated stimulation. We also provide evidence indicating that peroxynitrite is produced during in vitro immune activation, mainly by cells of the monocyte/macrophage lineage. Furthermore, immunohistochemical studies demonstrate the in vivo generation of nitrating species in human lymph nodes undergoing mild to strong immune activation. Our results point to a physiological role for ONOO- as a down-modulator of immune responses and also as key mediator in cellular and tissue injury associated with chronic activation of the immune system.
Protein tyrosine phosphatases (PTPs) contain an essential thiol in the active site which may be susceptible to attack by nitric oxide-derived biological oxidants. We assessed the effects of peroxynitrite, nitric oxide, and S-nitrosoglutathione on the activity of three human tyrosine phosphatases in vitro. The receptor-like T-cell tyrosine phosphatase (CD45), the non-receptor-like tyrosine phosphatase PTP1B, and leukocyte-antigen-related (LAR) phosphatase were all irreversibly inactivated by peroxynitrite in less than 1 s with IC(50) values of </=0.9 microM. PTP inactivation was also seen with equivalent concentrations of peroxynitrite generated by SIN-1, indicating that bolus peroxynitrite and cogeneration of superoxide and nitric oxide were equipotent. Rate constants for peroxynitrite-mediated PTP inactivation were determined by competition with cysteine and were among the fastest rates yet seen for reaction of peroxynitrite with any biological molecules. The bimolecular reaction rates for CD45, LAR, and PTP1B were 2.0 x 10(8), 2.3 x 10(7), and 2.2 x 10(7) M(-1) s(-1), respectively. Inactivation by peroxynitrite was essentially irreversible as incubation with dithiothreitol (DTT) restored less than 10% of the original phosphatase activity. Prolonged treatment with 0.4 mM DETA NONOate, which generated a steady-state concentration of 2 microM nitric oxide, was only slightly inhibitory. S-Nitrosoglutathione (1.0 mM) inhibited PTPs by approximately 50% after 30 min and the inhibition was completely reversed by DTT. Nitrotyrosine immunoblots of peroxynitrite-treated PTP1B revealed that peroxynitrite completely inactivated PTP1B prior to the appearance of protein tyrosine nitration. Peroxynitrite anion is structurally similar to phosphate anion both in terms of molecular diameter and charge. Thus, the extreme vulnerability of these PTPs to peroxynitrite-mediated inactivation is consistent with attraction of peroxynitrite anion to the active site and subsequent oxidation of the essential thiolate. These findings suggest that any PTP possessing the CXXXXXR active-site sequence could potentially be inactivated by peroxynitrite in vivo resulting in a net increase in tyrosine phosphorylation and profound effects on phosphotyrosine-dependent signaling cascades.
Tyrosine nitration is a covalent posttranslational protein modification derived from the reaction of proteins with nitrating agents. Protein nitration appears to be a selective process since not all tyrosine residues in proteins or all proteins are nitrated in vivo. To investigate factors that may determine the biological selectivity of protein tyrosine nitration, we developed an in vitro model consisting of three proteins with similar size but different three-dimensional structure and tyrosine content. Exposure of ribonuclease A to putative in vivo nitrating agents revealed preferential nitration of tyrosine residue Y(115). Tyrosine residue Y(23) and to a lesser extent residue Y(20) were preferentially nitrated in lysozyme, whereas tyrosine Y(102) was the only residue modified by nitration in phospholipase A(2). Tyrosine Y(115) was the residue modified by nitration after exposure of ribonuclease A to different nitrating agents: chemically synthesized peroxynitrite, nitric oxide, and superoxide generated by SIN-1 or myeloperoxidase (MPO)/H(2)O(2) plus nitrite (NO(-2)) in the presence of bicarbonate/CO(2). The nature of the nitrating agent determined in part the protein that would be predominantly modified by nitration in a mixture of all three proteins. Ribonuclease A was preferentially nitrated upon exposure to MPO/H(2)O(2)/NO(-2), whereas phospholipase A(2) was the primary target for nitration upon exposure to peroxynitrite. The data also suggest that the exposure of the aromatic ring to the surface of the protein, the location of the tyrosine on a loop structure, and its association with a neighboring negative charge are some of the factors determining the selectivity of tyrosine nitration in proteins.
Peroxynitrite (ONOO-), a potent oxidizing and nitrating species, has been linked to covalent modifications of biomolecules in a number of pathological conditions. In S. cerevisiae, a model eukaryotic cell system, ONOO- was found to be more potent than hydrogen peroxide in oxidizing thiols, inducing heat shock proteins (Hsp70) and enhancing the ubiquitination of proteins. As identified by microsequence analysis following immunoprecipitation with anti-nitrotyrosine antibodies, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was especially susceptible to nitration by ONOO- in yeast cells. The activity of this enzyme was strongly inhibited upon steady-state exposure of the cells to low doses of ONOO- in yeast and in cultured rat astrocytes. Thus, ONOO- is a potent stressor in yeast capable of inducing oxidative damage and protein nitration, with GAPDH being a preferential target protein that is efficiently inactivated.