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to the phenolic hydroxyl group) leading to protein tyrosine nitration (PTN) [5]. Protein tyrosine nitration exhibits a certain degree of
selectivity and not all tyrosine residues are nitrated since nitration may rather depend on the residue’s accessibility to solvents. Neither
the abundance of a protein nor the number of tyrosine residues in a given protein can help us predict whether it is a target for PTN [2,3,6].
For example, human serum albumin (HSA) is less extensively nitrated than other plasma proteins, although being the most abundant
plasma protein [6]. While HSA has 18 tyrosine residues, an in vitro study of peroxynitrite-mediated PTN showed that only two tyrosine
residues are particularly susceptible to nitration [7].
The reactivity of a tyrosine residue might also depend on the nature of the reactive species [2]. While peroxynitrite (ONOO - ) and
tetranitromethane (TNM) nitrate certain proteins [8], there are differences in PTN patterns in other proteins [9-11]. The nitration of
protein tyrosine residues could dramatically change protein structure and conformation and subsequently alter their function [12-15].
Tyrosine nitration sites are localized within specific functional domains of nitrated proteins [16]. For example, a strong inhibition of the
catalytic activity of manganese-superoxide dismutase (MnSOD) by peroxynitrite-mediated PTN has been reported and explained by
nitration of the essential tyrosine residue [17]. The inactivation of human MnSOD by peroxynitrite is caused by exclusive nitration of
tyrosine 34 (Tyr34) to 3-nitrotyrosine [18].
Role of Free Radical Species in Tyrosine Nitration
Nitrogen dioxide, nitrous acid, nitryl chloride, and certain peroxidases [19] 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 [20], 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 nitrates the tyrosine residues in the presence of hydrogen peroxide
(H 2
O 2
) [21]. Therefore, tyrosine nitration is based on the generation of nitrogen dioxide radicals (NO 2
) by various hemoperoxydases in
the presence of H 2
O 2
and nitrite [22-24]. Other plausible reactions are based on (i) the interaction of nitric oxide with a tyrosyl radical,
(ii) the direct action of nitrogen dioxide, (iii) the formation of nitrous acid by acidification of nitrite, (iv) the oxidation of nitrite by
hypochlorous acid to form nitryl chloride (NO 2
Cl), (v) the action of acyl or alkyl nitrates or (vi) the action of metal nitrates [1,25,26].
Hence, nitrotyrosine is therefore likely not a footprint for peroxynitrite alone but more generally a marker of nitrative stress.
Nitrating Agent Proteins nitrated References
Hemoperoxidases (myeloperoxidase, MPO
peroxidase) – MPO/H 2
O 2
system
Nitrite (NO 2-
)
Nitroprusside (nitropress)
Peroxynitrite (ONO 2–
)
Tetranitromethane (TNM)
lipoproteins
hemoglobin
p65 (NF-κB)
human serum albumin, creatine kinase, cytochrome c, hemoglobin,
NADH dehydrogenase, succinate dehydrogenase, H2A histone protein
bovine serum albumin, human serum albumin
Table 1: Factors or agents that lead to the formation of tyrosine nitrated proteins.
Tsikas (2012), Abello et al (2009).
The reactive nitrogen species, peroxynitrite, is an important nitrating agent in vivo. However, it is not the only source of 3-NT
formation in vivo. 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 [27,28]. However, later studies demonstrated that some
hemoproteins such as hemoglobin and myoglobin could catalyze NaNO 2
/H 2
O 2
-dependent nitration of tyrosine to yield 3-NT [29-31].
The possible underlying mechanism is that hemoproteins catalyze the oxidation of nitrite to nitrogen dioxide (NO 2
) which reacts with
tyrosine radical (Tyr . ) to form 3-NT [27,32,33].
Peroxynitrite and related reactive nitrogen species are capable of both oxidation and nitration of the aromatic side-chains of tyrosine
and tryptophan in proteins [34,35], 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 [36].
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)
[36]. In addition to the well known or standard 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 [37]. 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 [38].
Carbon dioxide/bicarbonate (1.3 and 25 mM in plasma, respectively) [39] strongly influence peroxynitrite-mediated reactions [39-
43] 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. Carbon dioxide reacts
with peroxynitrite to form the nitrosoperoxycarbonate anion (ONOOCO 2-
), which subsequently rearranges to form the nitrocarbonate
anion (O 2
NOCO 2-
). The latter is considered to be the direct oxidant of peroxynitrite-mediated reagents in biological environments [43].
Peroxynitrite-mediated tyrosine nitration is also accelerated in the presence of transition metal ions, either in free form (Cu 2+ , Fe 3+ , Fe 2+ )
or as complexes involving protoporphyrin IX (heme) or certain chelators - ethylene diamine tetraacetic acid (EDTA) [15,16,44]. Hence,
metal ions catalysis plays an important role in the nitration of protein residues in proteins [45].
Role of Protein Tyrosine Nitration in Pathological Conditions
A variety of proteins are nitrated at tyrosine residues both in vitro and in vivo by the reaction with peroxynitrite (Table 2) [46-49]. These
include, for instance, glutamine synthase [50], cdc2 kinase [51], bovine serum albumin (BSA) [52], MnSOD [53], phosphatidylinositol
3-kinase (PI3K) [54], and tyrosine hydroxylase [55]. 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 (Figures 2 and 3). Synovial fluid from patients with rheumatoid arthritis (RA) was found to contain a
higher concentration of NT-carrying immunoglobulin G (IgG) as compared to osteoarthritic (OA) specimens [56,57]. Peroxynitrite
reacts with several amino acids such as tyrosine, phenylalanine and histidine that are modified through intermediary secondary species
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