www.esciencecentral.org/ebooks Advances in Protein Chemistry Edited by Ghulam Md Ashraf Ishfaq Ahmed Sheikh Editors Dr. Ghulam Md Ashraf (Email: [email protected]) Dr. Ishfaq Ahmed Sheikh (Email: [email protected]) King Fahd Medical Research Center King Abdulaziz University, P.O. Box 80216 Jeddah, Saudi Arabia Tyrosine Nitrated Proteins: Biochemistry and Pathophysiology Haseeb Ahsan* Department of Biochemistry, Faculty of Dentistry, Jamia Millia Islamia (A Central University), Okhla, New Delhi – 110025, India *Corresponding author: Dr. Haseeb Ahsan, Department of Biochemistry, Faculty of Dentistry, Jamia Millia Islamia (A Central University), Okhla, New Delhi – 110025, India, E-mail: drhahsan@ gmail.com Abstract The free radical-mediated damage to proteins results in the modification of amino acid residues, cross-linking 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). 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. 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. Formation of tyrosine nitrated proteins is considered to be a post-translational modification with important pathophysiological consequences and is one of the markers of nitrosative stress that have been reported in neurodegeneration, inflammatory and other pathological conditions. Introduction Most proteins contain tyrosine residues with a natural abundance of about 3% [1]. Tyrosine (one letter abbreviation, Y; three letter abbreviation, Tyr; also known as 4-hydroxyphenylalanine) is a non-essential amino acid and a member of the aromatic amino acid group. Tyrosine is mildly hydrophilic, a characteristic feature that is explained by the rather hydrophobic aromatic benzene ring carrying a hydroxyl group. As a consequence, tyrosine is often surface-exposed in proteins (only 15% of tyrosine residues are buried inside a protein) and therefore should be available for modification such as nitration by various factors [2-4]. Tyrosine can be modified by the addition of a nitro group (-NO2) in vivo with several agents resulting in the formation of 3-nitrotyrosine (3-NT) or tyrosine nitrated proteins (Figure 1). 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 + + NH3 NH3 COO- COO- RNS NO2 OH OH Tyrosine Nitrotyrosine (Tyr) (3-NT) Tyrosine decarboxylase Tyramine oxidase metabolism metabolism COO- COO- RNS NO2 OH OH p-hydroxyphenylacetic acid 3-nito-4-hydroxyphenylacetic acid (NHPA) (PHPA) OMICS Group eBooks Figure 1: Metabolism of tyrosine leading to the formation of products: p-hydroxyphenylacetic acid and 3-nitro-4-hydroxyphenylacetic acid (adapted 003 from Mani et al., 2003). 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 (NO2-), 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 (H2O2) [21]. Therefore, tyrosine nitration is based on the generation of nitrogen dioxide radicals (NO2) by various hemoperoxydases in the presence of H2O2 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 (NO2Cl), (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 lipoproteins peroxidase) – MPO/H2O2 system - Nitrite (NO2 ) hemoglobin Nitroprusside (nitropress) p65 (NF-κB) Tsikas (2012), Abello et al (2009). human serum albumin, creatine kinase, cytochrome c, hemoglobin, Peroxynitrite (ONO –) 2 NADH dehydrogenase, succinate dehydrogenase, H2A histone protein Tetranitromethane (TNM) bovine serum albumin, human serum albumin Table 1: Factors or agents that lead to the formation of tyrosine nitrated proteins. The reactive nitrogen species, peroxynitrite, is an important nitrating agentin 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 NaNO2/H2O2-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 (NO2) 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 (ONOOCO2 ), which subsequently rearranges to form the nitrocarbonate - anion (O2NOCO2 ). 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 (Cu2+, Fe3+, Fe2+) 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