3-Nitrotyrosine: A biomarker of nitrogen free radical species modified proteins in systemic autoimmunogenic conditions

Human Immunology 74 (2013) 1392–1399

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3-Nitrotyrosine: A biomarker of nitrogen free radical species modified proteins in systemic autoimmunogenic conditions Haseeb Ahsan ⇑ Department of Biochemistry, Faculty of Dentistry, Jamia Millia Islamia, New Delhi, India

a r t i c l e

i n f o

Article history: Received 7 February 2013 Accepted 7 June 2013 Available online 15 June 2013

a b s t r a c t The free radical-mediated damage to proteins results in the modification of amino acid residues, crosslinking of side chains and fragmentation. L-Tyrosine and protein bound tyrosine are prone to attack by various mediators and reactive nitrogen intermediates to form 3-nitrotyrosine (3-NT). Activated macro phages produce superoxide (O 2 ) and NO, which are converted to peroxynitrite ONO2 . 3-NT formation is also catalyzed by a class of peroxidases utilizing nitrite and hydrogen peroxide as substrates. Evidence supports the formation of 3-NT in vivo in diverse pathologic conditions and 3-NT is thought to be a relatively specific marker of oxidative damage mediated by peroxynitrite. Free/protein-bound tyrosines are attacked by various RNS, including peroxynitrite, to form free/protein-bound 3-NT, which may provide insight into the etiopathogenesis of autoimmune conditions. The formation of nitrotyrosine represents a specific peroxynitrite-mediated protein modification; thus, detection of nitrotyrosine in proteins is considered as a biomarker for endogenous peroxynitrite activity. The peroxynitrite-driven oxidation and nitration of biomolecules may lead to autoimmune diseases such as systemic lupus. The subsequent release of altered proteins may enable them to act as antigen-inducing antibodies against self-proteins. Hence, tyrosine nitrated proteins can act as neoantigens and lead to the generation of autoantibodies against self proteins in various autoimmune disorders. Ó 2013 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Role of free radicals in tyrosine nitration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Formation of 3-NT in pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Autoimmunity: anti-tyrosine nitrated protein antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Tyrosine (Y, Tyr, 4-hydroxyphenylalanine) is a non-essential amino acid and a member of the aromatic amino acid group (the others being phenylalanine and tryptophan). Most proteins contain tyrosine residues with a natural abundance of about 3% [1]. Tyrosine is mildly hydrophilic, a characteristic feature that

Abbreviations: SLE, systemic lupus erythematosus; 3-NT, 3-nitrotyrosine; NO, nitric oxide; O 2 , superoxide anion; RNS, reactive nitrogen species; RA, rheumatoid arthritis; OA, osteoarthritis. ⇑ Address: Department of Biochemistry, Faculty of Dentistry, Jamia Millia Islamia (A Central University), Okhla, New Delhi – 110025, India. E-mail address: [email protected]

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is explained by the rather hydrophobic aromatic benzene ring carrying a hydroxyl group (Fig. 1A). As a consequence, tyrosine is often surface-exposed in proteins (only 15% of tyrosine residues are buried inside a protein) and is therefore available for modification such as nitration by various factors [2–4], resulting in the formation of 3-nitrotyrosine (3-NT) or tyrosine nitrated proteins (Fig. 1B). 3-NT {(2-Amino-3-(4-hydroxy-3-nitrophenyl) propanoic acid)} is a post-translational modification in proteins occurring through the action of a nitrating agent resulting in the addition of a NO2 group (in ortho position to the phenolic hydroxyl group) leading to protein tyrosine nitration (PTN) [5] (Fig. 2A and 2B). An important feature of PTN is the fact that it is a stable posttranslational modification and does not occur at random. Neither

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and subsequently alter their function [9–11]. Tyrosine nitration sites are localized within specific functional domains of nitrated proteins [12]. For example, nitration of Rho-GTPase-activating protein at 550Y affects signal transduction pathways mediated by Rho-GTPase [12]. Fig. 1A. Molecular structure of tyrosine (mol wt 181.19 g/mol, C9H11NO3).

1.1. Role of free radicals in tyrosine nitration

Fig. 1B. Molecular structure of 3-nitrotyrosine (mol wt 226.19 g/mol, C9H10N2O5).

Fig. 2A. Nitration of L-tyrosine to 3-nitrotyrosine. The nitrating agents are peroxynitrite (ONOO), nitryl chloride (NO2Cl), nitric oxide (NO), nitrous acid (HNO2), nitric acid (HNO3), nitrite NO 2 , hypochlorous acid (HOCl), nitrogen dioxide (NO2).

the abundance of a protein nor the tyrosine residues in a given protein can predict whether it is a target for PTN [2,3,6]. Not all tyrosine residues in a protein are available for nitration, which may rather depend on its accessibility to solvents. For example, human serum albumin (HSA), despite being the most abundant plasma protein, is a target for tyrosine nitration, but, is less extensively nitrated than other plasma proteins [6]. While HSA contains 18 tyrosine residues, an in vitro study of peroxynitrite-mediated PTN showed that only two tyrosine residues are particularly susceptible to nitration [7]. Protein tyrosine nitration exhibits a certain degree of selectivity, and not all tyrosine residues are nitrated [8]. The nitration of protein tyrosine residues could dramatically change protein structure and conformation

The reactive nitrogen species, peroxynitrite (ONOO), is an important nitrating agent in vivo. However, it is not the only source of 3-NT formation in vivo. Nitrogen dioxide, nitrous acid, nitryl chloride, and certain peroxidases [13] derived from inflammatory cells can mediate the nitration of tyrosine to form 3-NT (Table 1). For example, nitrite NO 2 , a primary autoxidation product of NO [14], is further oxidized to form nitrogen dioxide by the action of peroxidases, e.g., myeloperoxidase and eosinophil peroxidase, heme proteins that are abundantly expressed in activated leukocytes. The resulting nitrogen dioxide generated nitrates the tyrosine residues in the presence of hydrogen peroxide (H2O2) [15]. Therefore, tyrosine nitration is based on the generation of nitrogen dioxide radicals ðNO2 Þ by various hemoperoxydases in the presence of H2O2 and nitrite [16–18]. Other plausible reactions are based on (a) the interaction of nitric oxide with a tyrosyl radical, (b) the direct action of nitrogen dioxide, (c) the formation of nitrous acid by acidification of nitrite, (d) the oxidation of nitrite by hypochlorous acid to form nitryl chloride (NO2Cl), (e) the action of acyl or alkyl nitrates or (f) the action of metal nitrates [1,19,20]. Hence, nitrotyrosine is likely not a footprint for peroxynitrite alone but more generally a marker of nitrative stress. Initially, 3-NT was thought to be a biomarker of the existence of peroxynitrite, and protein tyrosine nitration was believed to be the biomarker of peroxynitrite formation in biological systems [21,22]. However, later studies demonstrated that some hemoproteins such as hemoglobin and myoglobin could catalyze NaNO2/H2O2-dependent nitration of tyrosine to yield 3-NT [23–25]. The possible underlying mechanism is that hemoprotein catalyzes the oxidation of nitrite to nitrogen dioxide (NO2) which reacts with tyrosine radical (Tyr.) to form 3-NT [21,26,27]. The reactivity of a tyrosine residue might thus also depend on the nature of the reactive species [2]. While peroxynitrite and tetranitromethane (TNM) nitrate certain proteins [28], there are differences in PTN patterns in other proteins [29–31]. A strong inhibition of the catalytic activity of

Fig. 2B. Metabolism of 3-nitrotyrosine leading to the formation of 3-nitro-4-hydroxyphenylacetic acid (adapted and modified from Mani et al. [105]).

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Table 1 Agents that cause tyrosine nitration in proteins. Nitrating agent

Proteins nitrated

Hemoperoxidases (such as myeloperoxidase, MPO peroxidase) – MPO/H2O2 Nitrite NO 2 Nitroprusside (nitropress) Peroxynitrite ONO 2

Lipoproteins

Tetranitromethane (TNM)

Hemoglobin p65 (NF-kB), Human serum albumin, creatine kinase, cytochrome c, hemoglobin, NADH dehydrogenase, Succinate dehydrogenase, H2A histone protein Bovine serum albumin, human serum albumin

Table 2 Peroxynitrite-induced nitration of proteins in various pathological conditions.a Protein(s) modified

Pathological indication

Actin protein Apolipoprotein A1 (ApoA1)

Endothelial dysfunction Inflammation, cardiovascular diseases Atherosclerosis Inflammation, ischemia

Apolipoprotein B100 (ApoB100) LDL ATP synthase complex I (FoF1 complex, F1-ATPase) Creatine kinase Cytochrome c Cytochrome P450 (2B6, 2E1) Glutamine synthase Glyceraldehy 3 phosphate dehydrogenase (GAPDH) Manganese superoxide dismutase (MnSOD) NADH dehydrogenase (NADH ubiquinone oxidoreductase, Complex I) Phosphorylase b Sarcoplasmic reticulum Ca2+ ATPase (SERCA2, calcium pump 2) Succinate dehydrogenase (complex II, succinate-coenzyme Q reductase) Tau protein

Myocardial infarction Inflammation, ischemia Various diseases Liver disease Cardiovascular, neurological diseases Neurodegenerative diseases Ischemia reperfusion Ageing Myocardial infarction Ischemia Alzheimer’s disease

a

Adapted and modified from Souza et al. [71], Yeo et al. [8], Tsikas [106], Peluffo and Radi [21], Lee et al. [107].

manganese-superoxide dismutase (MnSOD) by peroxynitrite-mediated PTN has been reported and explained by nitration of the essential tyrosine residue [32]. The inactivation of human MnSOD by peroxynitrite is caused by exclusive nitration of tyrosine 34 (Tyr34) to 3-nitrotyrosine [33]. A variety of proteins are nitrated at tyrosine residues both in vitro and in vivo by the reaction with peroxynitrite (Table 2) [34–37]. These include, glutamine synthase [38], cdc2 kinase [39], bovine serum albumin (BSA) [40], MnSOD [41], phosphatidylinositol 3-kinase (PI3K) [42], and tyrosine hydroxylase [43]. A proteomic approach has been able to identify more than 40 NT-carrying proteins that become modified as a consequence of inflammatory responses [5]. The formation of 3-NT proteins has been detected histochemically in inflamed or infected tissues (Fig. 3). Synovial fluid from patients with rheumatoid arthritis was found to contain a higher concentration of NT-carrying IgG as compared to osteoarthritic patients [44,45]. Peroxynitrite reacts with several amino acids such as tyrosine, phenylalanine and histidine that are modified through intermediary secondary species [46–49]. Protein sulfhydryls [50] and tyrosyl residues are the principal targets of peroxynitrite in proteins [51,52]. Nitration is maximal at physiological pH (pH 7.4), and its yield decreases under more acidic or basic conditions [52,53]. Carbon dioxide/bicarbonate (1.3 and 25 mM in plasma, respectively) [56] strongly influence peroxynitrite-mediated reactions [54–58]

Fig. 3. Immunohistochemistry of protein-bound 3-nitrotyrosine. Nitrotyrosine staining of a human lymph node undergoing nonspecific immune activation is shown. The arrowhead indicates the highest 3-nitrotyrosine immunoreactivity in macrophages. The tissue sections were probed with anti-nitrotyrosine polyclonal antibodies (adapted and modified from Radi et al. [108]).

and enhance nitration of aromatic rings as in tyrosine. They can also promote nitration in the presence of antioxidants (such as uric acid, ascorbate and thiols), which normally inhibit nitration, while partially inhibiting the oxidation of thiols. Peroxynitrite-mediated tyrosine nitration is also accelerated in the presence of transition metal ions, either in free form (Cu2+, Fe3+, Fe2+) or as complexes involving protoporphyrin IX (heme) or certain chelators–ethylene diamine tetraacetic acid (EDTA) [11,12,59]. Hence, metal ion catalysis plays an important role in the nitration of protein residues in proteins [60]. Peroxynitrite and related reactive nitrogen species are capable of both oxidation and nitration of the aromatic side-chains of tyrosine and tryptophan in proteins [61,62], resulting in a condition known as ‘‘nitrosative stress’’. Reactive oxygen species (ROS) and reactive nitrogen species (RNS)-mediated damage in the central nervous tissues may reflect an underlying neuroinflammatory process [63]. Protein damage that occurs under conditions of oxidative stress may represent direct oxidation of protein side-chains by ROS and/or RNS or adduction of secondary products of oxidation of sugars (glycoxidation), or polyunsaturated fatty acids (lipid peroxidation) [63]. In addition to well known or classical reactive oxygen and nitrogen species, oxidative damage to proteins can occur due to alternate oxidants (e.g., HOCl) and circulating oxidized amino acids such as tyrosine radical generated by metalloenzymes such as, myeloperoxidase [64]. The accumulation of oxidized protein is a complex function of the rates of ROS formation, antioxidant levels, and the ability to proteolytically eliminate ion of oxidized forms of proteins [65]. Nitration of tyrosine residues can profoundly alter protein structure and function, suggesting that protein nitration may be fundamentally related to and predictive of oxidative cell injury. The subsequent release of altered proteins may enable them to act as antigens inducing antibodies against self-proteins. The biological significance of tyrosine nitration supports the formation of 3-NT in vivo in diverse pathologic conditions and 3-NT is thought to be a relatively specific marker of oxidative damage mediated by peroxynitrite and other nitrogen free radical species. The immunoreactivity of 3-NT has been reported in several human pathological conditions [66]. Free/protein-bound tyrosine are attacked by various RNS, including peroxynitrite, to form free/protein-bound 3-NT, which may provide insight into the mechanism

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of severe disease activity as seen in lupus patients. In addition, numerous other disease states using non-human models have been shown to involve the formation of 3-NT [67]. 1.2. Formation of 3-NT in pathophysiology Numerous studies have shown the presence of 3-nitrotyrosine in several pathologic conditions. Elevated levels of 3-nitrotyrosine have been reported is various human pathologies such as atherosclerosis, multiple sclerosis, Alzheimer’s disease, Parkinson’s disease and its animals models, cystic fibrosis, asthma, lung diseases, myocardial malfunction, stroke, amyotrophic lateral sclerosis, chronic hepatitis, cirrhosis, diabetes, etc. [68]. Formation of 3-nitrotyrosine in proteins is considered to be a post-translational modification with important pathophysiological consequences and is one of the early markers of nitrosative stress that have been revealed in multiple sclerosis. Elevated contents of nitrates, nitrites, and free nitrotyrosine were found in the cerebrospinal fluid of subjects and have been proposed as functional biomarkers of neurodegeneration [69]. Approximately 1–10 residues of tyrosine per 100,000 (10–100 lmol 3-NT/mol of tyrosine) are found to be nitrated in plasma proteins under inflammatory conditions such as in cardiovascular disease [70], although upto 10 times more 3-NT can be detected in tissues [13]. Studies have found a strong correlation between 3-NT plasma levels and coronary artery disease (CAD), identifying the elevated levels of 3-NT as an emerging cardiovascular risk factor [71] (Table 3). Nitric oxide is known to participate as a cytotoxic effector molecule or a pathogenic mediator when overexpressed by either inflammatory stimuli-induced nitric oxide synthase (iNOS) or over stimulation of the constitutive forms of NOS (eNOS). The autologous proteins may become immunogenic if they are structurally modified post-translationally under physiological and pathological conditions. These chemical modifications include transglutamination, deamidation, glycosylation, oxidation, nitration and proteolytic cleavage. The consequence of these protein modifications may be generation or unmasking of new antigenic epitopes, which will stimulate relevant B cells and/or T cells, thus leading to the breakdown or bypass of tolerance. Among the protein modifications mentioned, protein tyrosine nitration is widely recognized as a hallmark of inflammation that is associated with the up-regulation of iNOS and is not affected by exogenous sources of nitrate/ nitrite (NO3/NO2) or serum thiols [72–75]. 1.3. Autoimmunity: anti-tyrosine nitrated protein antibodies The presence of the nitro-group in a tyrosine residue can generate an immunological response [76]. In the plasma from patients with post-traumatic acute lung injury, immunoglobulins against 3-nitrotyrosine were detected showing that the nitroxidative stress was able to elicit an immunological response in the endogenous nitrated proteins generated during lung injury [77]. Nitrated peptide generated by the antigen presenting cells during antigen processing also stimulated the CD4 T response [78,79]. These findings raise the possibility that nitrated epitopes of autologous proteins could be linked to the etiopathogenesis of some autoimmune diseases [71]. It has been suggested that alteration in amino acid structure or

Table 3 Level of 3-nitrotyrosine measured in different human samples.a

a

Samples

Level of 3-NT

Pathological condition

Human plasma Synovial fluid

31.6 ± 6 nmol/L 2.8 ± 0.84 nmol/L 0–1.2 lmol/L

Oxidative stress Rheumatoid arthritis

Adapted and modified from Duncan [68].

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sequence may generate neoepitopes on self-proteins, leading to an immune attack. The modifications may generate or mask antigenic epitopes and stimulate relevant B cells and/or T cells, leading to a breakdown or bypass of tolerance [80]. Studies on the recognition of tyrosine nitrated proteins by Tcells shows that PTN of a tyrosine in a T-cell receptor (TCR) contact position may result in the formation of an immunogenic neoepitope [81,78]. Moreover, it has been shown that nitration of tyrosines located in the non-TCR-contact positions can have an indirect yet major impact on stimulation of the immune system by affecting interactions of the TCR with the peptide antigen loaded major histocompatibility complex (MHC) [72]. A variety of post-translationally modified (including nitrated) proteins have been shown to accumulate in apoptotic or inflamed tissues [82]. Hence, the accumulation of nitrotyrosine-containing proteins in tissues that appear as foreign to the immune system might induce an autoimmune response and sustain a chronic inflammatory reaction [78]. Elevated levels of anti-nitrotyrosine antibodies have been measured in the synovial fluid of patients with rheumatoid arthritis and osteoarthritis [73], serum of patients with systemic lupus erythematosus [83] or after acute lung injury [84]. PTN of endogenous proteins may not only trigger harmful, abnormal immune responses but also hinder beneficial, normal responses due to defective T-cell receptor (TCR) recognition [78] or due to PTN of the TCR itself. Cells of the immunoregulatory network produce both superoxide anion and nitric oxide during oxidative burst triggered by inflammation. Nitric oxide and superoxide may combine to generate peroxynitrite. The peroxynitrite-driven oxidation and nitration of biomolecules (protein, lipid, DNA) may lead to lupus-like autoimmunity and age-related diseases [85,86]. Furthermore, nitration of tyrosine residues can profoundly alter protein function, suggesting that protein nitration may be fundamentally related to, and be predictive of, oxidative cell injury. The subsequent release of altered proteins may enable them to act as antigens inducing antibodies against these modified self-proteins (Table 4). Autoantibodies targeted against intracellular proteins and nucleic acids are the serological hallmark of the systemic rheumatic diseases, such as systemic lupus erythematosus (SLE), progressive systemic sclerosis (PSS), Sjogren’s syndrome (SS), mixed connective tissue disease (MCTD) and polymyositis (PM). Each one of these diseases is characterized by unique autoantibodies. SLE sera screened by direct binding and competition ELISA and showed stronger binding to nitrated poly L-tyrosine when compared to native poly L-tyrosine and nDNA [83]. Several lines of evidence indicate that RNS maybe important in the pathogenesis of rheumatoid arthritis, osteoarthritis and SLE. Peripheral blood mononuclear cells from RA patients have increased expression of iNOS and enhanced formation of NO that correlates with disease activity. In addition, NO has been shown to be a key mediator of apoptosis within the rheumatic/arthritic joints and an important regulator of the Th1/Th2 balance in autoimmune diseases [87]. Systemic lupus erythematosus is the prototypical systemic chronic autoimmune disease, characterized by diverse clinical manifestations and production of multiple autoantibodies. Common long-term complications of SLE include damage to the musculoskeletal, neuropsychiatric, renal, and cardiovascular systems [73,88]. It has also been found that serum 3-nitrotyrosine level is elevated among patients with SLE [75,89]. Several studies have indicated a correlation between serum nitrate/nitrite level and disease activity in SLE [83]. The data of Khan and Siddiqui [73] provides evidence in support of earlier suggestion that NO and its intermediates maybe a mediator in chronic inflammatory joint disease. Their results in the case of SLE patients are consistent with an increased extent of protein nitration in the serum samples. Since the increase in anti-3-nitrotyrosine antibodies is due to

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H. Ahsan / Human Immunology 74 (2013) 1392–1399 Table 4 Role of tyrosine nitration in autoimmune phenomenon.* Molecular target

Nitrating agent

Probable mechanism

Pathological indication

Histone protein (H2A) Histone protein (H2A) Poly-tyrosine Serum proteins Synovial tissue proteins

Peroxynitrite Peroxynitrite Nitric oxide Nitric oxide Nitric oxide

Formation Formation Formation Formation Formation

SLE RA RA, OA, SLE SLE RA, OA

of of of of of

nitrotyrosine, dityrosine, carbonyls 3-NT 3-NT 3-NT nitrated (nitrotyrosine) proteins

SLE, systemic lupus erythematosus; RA, rheumatoid arthritis; OA, osteoarthritis. Adapted and modified from Ahmad and Ahsan [94]; [Epub ahead of print].

*

increase in concentration of 3-nitrotyrosine in the sera and synovial fluid samples, it can be believed that levels of 3-nitrotyrosine correlate with disease severity. The enhanced level of nitration detected in patients with higher level of disease activity suggests that the measurement of protein nitration may be a useful surrogate marker of disease activity [73]. SLE is a prototype autoimmune, multisystem and multifactorial disease characterized by the presence of auto-antibodies to a variety of nuclear antigens such as DNA and histones, as well as protein antigens and protein–nucleic acid complexes. SLE occurs mainly in females (9-fold higher). As with RA, the initial immunizing antigen(s) that drive the development of SLE are unknown, but characteristics of the immune response in SLE suggest that it is an antigen-driven process [90]. A wide variety of inflammatory lesions occur in the kidney, spleen, lungs, joints and other organs. Immune complex deposits in the synovium are associated with mild inflammation and cartilage destruction is seldom severe. The arthritis in SLE is described as non-destructive and nondeforming. Usually polyarthritis is more severe in later stages and may even resemble RA [73,91].

1

2

3

4

5

6

7

8

9

Fig. 4. Immunoblot analysis of 3-nitrotyrosine in the serum of SLE patients. Lane 1: nitrated bovine serum albumin; lanes 2–3: normal human sera; lanes 4–9: different SLE serum samples (adapted and modified from Khan et al. [92]).

Khan et al. [92] have shown that the serum 3-NT is elevated in patients with SLE. The findings are in agreement of previous studies indicating that in SLE patients there is an upregulation of iNOS and increased NO production has also been observed in human and murine disease. When 3-NT is evaluated with native dsDNA and native poly L-tyrosine, it binds more strongly with SLE subjects. It is likely that reactive nitrogen species penetrate the cellular membranes and in turn react with proteins, thereby resulting in their modification [93,94] (Fig. 4). Histones are small cationic proteins that bind DNA and remain confined to the nucleus. They are weak immunogens, probably because of their conserved nature. However, after apoptosis they may appear in the circulation as nucleosomes. Histones show strong immunogenicity after acetylation and nitration [95]. The incidence of autoantibodies against H1, H2A, H2B, H3, and H4 histones in the sera of SLE patients has been reported [96]. In addition, antinative DNA autoantibodies are commonly copresent with antihistone autoantibodies and may react with each of the five chromatin associated histones or their complexes [97]. Both DNA and histones are components of the nucleosome [98]. Each nucleosome is composed of a central tetramer of 2 molecules each of H3 and H4 histones, flanked by 2 dimers of histones H2A and H2B and surrounded by about 160bp of DNA. Histone H1 is located on the outside of the octamer complex, external to the nucleosome. Antinuclear antibodies in human and murine lupus can distinguish complex epitopes that result from the ordered interactions between histones and DNA [99,100]. Monoclonal antibodies to H2A–H2B–DNA (or to more complex nucleosome epitopes) have been isolated from spontaneously autoimmune mice [101,102]. The histone proteins show enhanced immunogenicity after acetylation and alterations in amino acid structure or sequence and can generate neo-epitopes on self proteins causing an immune attack. The oxidative and nitrative action of peroxynitrite confers

Fig. 5A. High Performance Liquid Chromatographic profile of 3-nitrotyrosine peak of normal human serum. The arrow indicates the absence of 3-nitrotyrosine in the control samples [80].

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Fig. 5B. High Performance Liquid Chromatographic profile of 3-nitrotyrosine peak of SLE serum. The arrow indicates the 3-nitrotyrosine peak in the SLE serum samples (adapted and modified from Dixit et al. [80]).

additional immunogenicity on H2A histone indicating a direct correlation between nitration and immunogenicity. Hence, immunization with peroxynityrite-modified H2A may produce polyspecific antibodies which can recognize and bind to both old and newly formed epitopes [103]. Peroxynitrite induces an array of modifications in H2A structure such as tyrosine nitration, protein carbonyl, dityrosine and crosslinking. Such structural modifications might favor polymerization of native epitopes of H2A histone into potent immunogenic neo-epitopes [80] (Figs. 5A and 5B). The nitrotyrosine-bearing epitopes on proteins for example, serum proteins may be effective in activating relevant T cells. Investigation into how immunological tolerance is disturbed by post-translationally modified self-proteins including nitrated proteins may lead to more profound understanding of the onset of autoimmune responses. The changes in self-proteins suggest either that nitration occurs in SLE patients at an elevated level or that nitration occurs in acute oxidative bursts, with inefficient repair of these lesions once they are formed. Approaches directed at inhibiting the oxidative modifications caused by reactive nitrogen species can open new avenues for the treatment of inflammatory, vascular and neurodegenerative diseases. Further studies are also required to determine the pathogenicity of RNS-mediated protein modifications in various autoimmune phenomena [104].

Acknowledgments H.A. also wishes to acknowledge those authors whose work may have been inadvertently not mentioned in the manuscript. This review article is dedicated to Professor Rashid Ali, former chairman, Department of Biochemistry, J.N. Medical College, Aligarh.

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