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Biochemistry of protein tyrosine nitration in cardiovascular pathology

Gonzalo Peluffo, Rafael Radi
DOI: http://dx.doi.org/10.1016/j.cardiores.2007.04.024 291-302 First published online: 15 July 2007


Several pathologies of the cardiovascular system are associated with an augmented production of nitric oxide and/or superoxide-derived oxidants and/or alteration in the antioxidant detoxification pathways that lead to nitroxidative stress. One important consequence of these reactive intermediates at the biochemical level is the nitration of protein tyrosines, which is performed through either of two of the relevant nitration pathways that operate in vivo, namely peroxynitrite and heme peroxidase-dependent nitration. Proteins nitrated at tyrosine residues have been detected in several compartments of the cardiovascular system. In this review a selection of nitrated proteins in plasma (fibrinogen, plasmin, Apo-1), vessel wall (Apo-B, cyclooxygenase, prostaglandin synthase, Mn-superoxide dismutase) and myocardium (myofibrillar creatine kinase, α-actinin, sarcoplasmic reticulum Ca2+ ATPase) are analyzed in the context of cardiovascular disease. Nitration of tyrosine can affect protein function, which could directly link nitroxidative stress to the molecular alterations found in disease. While some proteins are inactivated by nitration (e.g. Mn-SOD) others undergo a gain-of-function (e.g. fibrinogen) that can have an ample impact on the pathophysiology of the cardiovascular system. Nitrotyrosine is also emerging as a novel independent marker of cardiovascular disease. Pharmacological strategies directed towards inhibiting protein nitration will assist to shed light on the relevance of this post-translational modification to human cardiovascular pathology.

  • Free radicals
  • Nitric oxide
  • Nitration
  • Nitroxidative stress
  • Nitrotyrosine
  • Peroxynitrite

Time for primary review 49 days

1 Introduction

Tyrosine nitrated proteins1 constitute a widespread finding in the normal or diseased cardiovascular (CV) system. Specially, in the various compartments and tissues of the CV system, namely, intravascular space, vessel wall and myocardium, nitrated proteins have been detected with a variety of techniques (reviewed in [1] and [2]). The role of 3-NO2-Tyr as an emerging cardiovascular risk factor is underscored by recent work that shows a strong association between 3-NO2-Tyr levels and coronary artery disease (CAD) after correction for classical cardiovascular (CV) risk factors [3].

Nitrotyrosine accumulation reflects a loss of balance between oxidant formation and antioxidant defence mechanisms, formerly known as oxidative stress [4]. Nitration of biomolecules, such as proteins and lipids have gained great biological significance and are largely dependent on nitric oxide (NO)-derived oxidants [5]. This conceptualization led us to use the term “nitroxidative stress” to reinforce the concept that nitration is an oxidative modification itself and will be used herein within the scope of previous work [6,7].

Early after the discovery of NO [8], it became evident that due to its radical character, it could participate in free radical-mediated processes under circumstances where it is produced at high rates or upon its reactions with other intermediaries such as superoxide (O2) that augments the oxidant potency of the otherwise mildly reactive NO [9,10].

2 Impaired NO bioavailability during vascular oxidant formation

Nitric oxide, synthesized from the oxidation of l-arginine by nitric oxide synthase (NOS), is a major contributor to the normal homeostasis of the cardiovascular system. Physiological levels (nM range) of NO produced by the endothelial isoform of NOS (eNOS), are principal determinants of endothelium-dependent relaxation and regulator of vascular tone. Furthermore, NO inhibits platelet aggregation [10] adhesive molecules expression [11], and regulates cell proliferation and differentiation at the vascular wall [12]. A decrease in the bioavailability of NO is associated with a myriad of cardiovascular disease conditions, including atheromatosis, heart failure, sepsis, coronary artery disease (CAD), stroke and myocardial infarction, among others.

The above mentioned pathologies are associated with an increase in production of reactive oxygen species (ROS) in particular O2, the one electron reduction product of molecular oxygen. Sources of O2 include NAD(P)H oxidases (NOX), xanthine oxidase (XO), mitochondria and uncoupled NOS. Most importantly, endothelial cells, smooth muscle cells and cardiomyocytes are major sites of O2 production in the cardiovascular system. Extensive analysis of O2 production mechanisms by these enzymes, is beyond the scope of this review (see [13]).

Nitric oxide produced intracellularly at the endothelial cell readily diffuses out and exerts its action at the smooth muscle cell by activation of soluble guanylate cyclase (sGC). Under pathological conditions that have endothelial dysfunction, NO bioavailability is hampered either through a decrease in its production by the presence of endogenous NOS inhibitors such as asymmetric di-methyl-arginine (ADMA), by uncoupling of NOS due to tetrahydrobiopterin oxidation or by its oxidative inactivation via its reaction with O2. In this regard, several disease states have been shown to occur with elevated levels of ADMA including, hypertension, CAD and diabetes but will not be discussed herein [14].

Cellular and tissue defences against ROS include the enzymes superoxide dismutase (Mn-SOD, Cu/Zn-SOD, extracellular (EC)-SOD), catalase, glutathione peroxidase, peroxiredoxins and the non-enzymatic antioxidants, glutathione (GSH), thioredoxin, ascorbate, α-tocopherol and uric acid [15]. It is well documented that relatively low levels of ROS act physiologically at the signalling level, modulating proliferation, apoptosis [16] and gene expression through the activation of transcription factors such as NF-kappa-B and hypoxia-inducible-factor-1α (HIF) [17].

A relevant pathway that decreases NO levels and therefore inactivates its signalling activities, is the diffusion-controlled reaction with O2 (k≈1×1010 M−1 s−1) that gives rise to the powerful oxidizing and nitrating agent, peroxynitrite (ONOO) [18]. Peroxynitrite is a short living species (10 ms in vivo) [19] which can exert damage by direct reaction via one- or two-electron oxidation mechanisms with several molecules such as thiols, metal centres and notably, carbon dioxide (CO2) [5]. Peroxynitrite also yields secondary one-electron oxidants (hydroxyl radical (OH); nitrogen dioxide (NO2); carbonate radical (CO3)) which can damage lipids, DNA, participate in hydroxylation, oxidation and most importantly, nitration reactions [20]. When either O2 or NO levels increase, ONOO is formed in spite of the SOD-catalyzed dismutation of O2 (k=1×109) [21] and the diffusion of NO. It is important to emphasize that not only a potent oxidant such as ONOO is formed but also that the beneficial protective actions of NO (anti-inflammatory, anti-proliferative and anti-aggregant) are lost after the reaction with O2. Thus, a shift from signal transduction to oxidative pathophysiology occurs after the reaction of O2 with NO. Several issues must be considered in order to put in perspective the feasibility of this reaction in vivo due to the alternative targets for the precursors of ONOO, and of ONOO itself [22].

In addition to O2, other oxidative mechanisms that down regulate the biological actions of NO, involve heme-peroxidases such as myeloperoxidase (MPO) which are present at inflammation sites (e.g. atheroma plaque), and iron (non heme)-peroxidases for instance lipoxygenase (LPO), that can catalytically consume NO in the presence of hydrogen peroxide (H2O2) acting as NO-sinks and compromising its bioavailability [23]. Lastly, during lipid peroxidation processes that occur in pathological conditions both in biological membranes and lipoproteins, lipid peroxyl radicals can react fast in the lipid phase with NO in a radical–radical termination reaction giving rise to nitrolipids (LONO2) which may have biological activity [24].

3 Nitroxidative stress in vivo

The evidence for protein nitration in vivo is copious and solid [25] although there is some discussion on the main nitrating mechanisms involved. Originally, nitration was associated with the generation of ONOO only and it was considered as a totally specific footprint of this molecule [20]. Alternative mechanisms of nitration have been shown to take place in vivo (vide infra). In any case, every possible mechanism requires the concomitant presence of NO and NO-derived species (NO2, ONOO, NO2) and oxidants (O2, H2O2, CO3, oxo metal centres [Me(n+1)+]) [20].

Recently, the impact of intracellular O2 steady state (ss) concentrations on NO output from a hypothetical cell, have been modelled in silico [22]. The model considers a sustained intracellular source of NO such as activated eNOS in vascular endothelial cells, that will either diffuse out of the cell or react with intracellular O2 to yield ONOO (Fig. 1). Reactions of NO, O2 and ONOO with the most relevant intracellular targets were considered in the experimental model. At first glance, one will intuitively predict that an increase in the steady state concentration of O2 on the cell will bring NO concentration down due to the fast reaction between these two radicals. Paradoxically and counter intuitively, intracellular NO steady state concentrations are not significantly affected by an increase on intracellular O2. Since diffusion is a concentration-dependent process, the reaction of NO with O2 limits diffusion of NO out of the cell and reestablishes the intracellular steady state concentration of NO. Thus, the main effect of increasing intracellular and even extracellular O2 is to affect NO output and therefore NO bioavailability as a paracrine signalling molecule. We speculate that these findings imply that sGC in the arterial smooth muscle cell may “see” a much larger difference on NO concentration than the O2producing endothelial cell and explains how O2, at the NO production site, can control or alter its signalling properties at a distant action site. Fig. 1 shows three hypothetical scenarios and the effect of O2 levels on NO output of an endothelial cell.

Fig. 1

Regulation of NO output from the endothelium by O2. (A) Nitric oxide produced in the endothelial cell diffuses towards the smooth muscle cell, activates sGC and thus cGMP-dependent vessel relaxation. Physiologically produced O2 is detoxified by SOD although some escapes and reacts with NO to form ONOO that can participate in tyrosine nitration. In fact, low levels of nitrated proteins are found in normal conditions. (B) When endothelial O2 levels increase, for example after angiotensin I-dependent NOX activation or inactivation of SOD, NO output by the cell is hampered, affecting cGMP-dependent relaxation at the smooth muscle and other NO-dependent actions in the vasculature. In parallel, intracellular ONOO formation and tyrosine nitration increases, as observed in various pathologies. Importantly, the steady state intracellular NO concentration in the endothelial cell is minimally affected. (C) When an extracellular O2 source is present or EC-SOD is inactivated, O2 will react with NO diffusing to the smooth muscle cell and as a consequence ONOO will be formed in the extracellular compartment. In this scenario, alterations in vessel homeostasis and extracellular protein nitration will be observed but, again, the intracellular steady state NO concentration in the endothelial cell will be preserved. Colour shades in endothelial and smooth muscle cells represent NO concentration.

Finally, in spite of alternative fast reacting targets for intracellularly-produced ONOO such as thiols (GSH, thioredoxin) which are present at high concentrations in the cytosol, the fraction of ONOO that can form 3-NO2-Tyr, increases in parallel with O2 and/or NO production rates [22,26,27]. A similar mechanism but with extracellular generation of ONOO can be envisioned to account for extracellular protein nitration as observed in plasma and matrix protein components (Fig. 1). Heme-peroxidases released from activated leucocytes also participate in extracellular protein nitration. Therefore NO-derived oxidants impose a nitroxidative stress in the cardiovascular system.

4 Biochemical mechanisms of tyrosine nitration: the radical pathways

Biological nitration of tyrosine depends largely on free radical chemistry. There are two key nitration pathways that operate in vivo and that involve peroxynitrite and hemoperoxidase-catalyzed nitration (Fig. 2). Tyrosine nitration is a two-step process where the initial reaction is the oxidation of the aromatic ring of tyrosine to yield tyrosyl radical (Tyr) (oxidation step), which in turns adds NO2 (addition step) to yield 3-NO2-Tyr. The principal difference between both reaction pathways lies in the oxidation step that generates Tyr by a one-electron oxidation process [1,5].

Fig. 2

Principal tyrosine nitration pathways. The scheme represents the two main nitration pathways involved in tyrosine nitration in the cardiovascular system. Peroxynitrite through its reaction with CO2, present at high concentration in biological fluids, yields one-electron oxidants (CO3•− and NO2) that form tyrosyl radical (Tyr), the first (step 1) step en route to 3-NO2-Tyr. Compound I of myeloperoxidase, which is formed during the catalytical cycle of MPO can also oxidize Tyr to Tyr (step 1). NO2 derived either after the homolysis of ONOO or after the C-1-catalyzed oxidation of nitrite (NO2), adds to the Tyr in a fast radical–radical termination reaction to yield 3-NO2-Tyr (step 2). Although both ONOO and H2O2 fuel nitration pathways, is important to underscore that O2•− and NO are the usual precursors of both oxidants. Moreover NO2 is derived from the decomposition of NO or ONOO. The reactions of peroxynitrite with transition metal centres and tyrosine nitration are discussed elsewhere [5].

Peroxynitrite (pKa=6.8) is partially protonated at pH 7.4 to peroxynitrous acid (ONOOH) which after homolysis produces OH and NO2 in approximately 30% yields. Hydroxyl radical which is a potent oxidizing agent, reacts rapidly with tyrosine (1.2×1010 M−1 s−1) to form Tyr but in low yields (5%) since most of the OH adds to the tyrosine ring giving rise to hydroxylated products such as 3,4-di-hydroxy-phenylalanine (DOPA). Although ONOO per se represents a minor route for tyrosine nitration in vivo, its fast reaction with carbon dioxide (CO2), which is present at 1–2 mM concentration in biological fluids, catalyzes peroxynitrite-dependent nitration processes. Peroxynitrite reacts fast with CO2 to generate a nitroso-peroxocarboxylate adduct (ONOOCO2) which in turns undergoes homolysis to CO3 and NO2 in 35% yields. Contrary to what is observed for OH, CO3 radical is highly effective in oxidizing tyrosine to Tyr (Fig. 2). Therefore, the reaction with CO2, redirects ONOO towards a more effective nitration pathway, which is likely to be a predominant one in vivo (for a comprehensive review see [1,5]).

Heme-peroxidases such as MPO and eosinophil peroxidase are capable of catalyzing free and protein tyrosine nitration in the presence of H2O2 and nitrite (NO2). After reaction with H2O2, the heme allocated at the active site undergoes oxidation to Compound I (MPOFe4+) which is a strong oxidant that can perform the first step in the sequence that leads to 3-NO2-Tyr (formation of Tyr). Additionally, Compound I can oxidize NO2 to NO2 that adds to the Tyr forming 3-NO2-Tyr [28,29]. Most importantly, H2O2 in biological systems arises in a significant proportion from O2 and NO2 from NO (either as a direct oxidation product or from ONOO that can itself evolve to NO2 secondary to target molecule reactions).2

Mechanisms of tyrosine nitration have been typically studied in aqueous phases in spite of the presence of 3-NO2Tyr in transmembrane or hydrophobic domains of several proteins such as apolipoprotein-A I (apo AI), apolipoprotein-B (apo B) and sarcoplasmic reticulum Ca2+ ATPase (SERCA). Recently published data (reviewed in [1]) had shed light on tyrosine nitration in hydrophobic phases by using hydrophobic analogs of Tyr such as N-t-BOC-l-tyrosine tert-butyl ester (BTBE) incorporated to liposomes [30]. Lipid phases with polyunsaturated fatty acids are susceptible to lipid peroxidation processes with the formation of lipid-derived radicals that are likely to oxidize tyrosine to Tyr. Oxidants, such as NO2 that concentrate in hydrophobic phases, favour their reaction with Tyr in lipid-rich environments.

Another alternative pathway for 3-NO2-Tyr is the reaction of Tyr with NO that yields 3-NO2-Tyr via the intermediacy of 3-nitrosotyrosine and tyrosine iminoxyl radical. This path to 3-NO2-Tyr requires a two-electron oxidation of nitrosotyrosine that has been reported on transition metal-containing proteins such as COX [31]. Protein tyrosine nitration shows a certain degree of selectivity and not all tyrosine-containing proteins or tyrosine residues within a single protein are equally modified. The protein and environment-dependent factors that control site- and protein-specificity in tyrosine nitration processes have been recently reviewed [1,32].

5 Protein nitration in the cardiovascular system

We will analyze selected nitrated proteins from different compartments of the cardiovascular system (Table 1) based mainly on human in vivo data of pathological relevance. Identification of tyrosine nitrated proteins is a fertile research field with a large quantity of data becoming available mainly due to proteomics-based methods.

View this table:
Table 1

Selected nitrated proteins in the cardiovascular system

Nitrated proteinMethodologyMajor observationsRef
Plasma proteins
FibrinogenIP of fibrinogen; 3-NO2-Tyr determination by HPLC-ESI-MS-MS; Electron microscopy30% increase on nitrated fibrinogen in CAD; accelerated clot formation; fragile clot[37]
PlasminIP with anti-3-NO2-Tyr antibody of plasma proteins; WB to major plasma proteinsIncrease in nitration of plasmin and fibrinogen in smokers; in vitro inactivation by nitration of plasminogen[38,39]
Apo A-IIP with anti 3-NO2-Tyr antibody from plasma and apo-1 isolation from biopsies; proteomic methodology for protein identification; HPLC-ESI MS-MS for the determination of 3-NO2-Tyr and 3-Cl-TyrApo-1/MPO interaction; nitration and chlorination of Tyr192; two-fold increase of nitrated Apo-1 in CAD; preferential nitration of Apo-1 in plasma when compared to other nitrated plasmatic proteins[44,45]
Vessel wall
Apo BLDL purification by ultracentrifugation of human plasma and aortic lesion; 3-NO2-Tyr was determined after hydrolysis and derivatization by GC–MS90-fold concentration of nitrated LDL in aortic lesion when compared to plasma nitrated LDL[54]
COXIP of COX-1 from smooth muscle cells and from human atheroma plaques; WB and spectrophotometrical determination of nitrated COX-1; COX-1 EPR studies; trypsin digestion, HPLC purification and amino acid sequencingIn vitro inactivation by nitration of COX-1; identification of nitrated COX-1 in smooth muscle cells and in human atheroma plaques; identification of nitrated Tyr385 by NO and Tyr[62–64]
PGISIP of endothelial nitrated proteins; WB; immunohistochemistry; thermolysin digestion of nitrated PGIS and high resolution MS (FT-ICR) of nitrated peptidesNitration of PGIS at low ONOO (1 μM); IC50 for inactivation 100 nM; colocalisation of nitrated proteins and PGIS in the endothelium bovine coronary artery; specific nitration at Tyr430[65,67]
Mn-SODIn vitro nitration of human Mn-SOD by ONOO; ESI-MS analysis of digested SOD; amino acid sequencing; IP of Mn-SOD of aged rat aorta; WB; immunoelectron microscopy anti 3-NO2-Ty; development of specific Ab against nitrated SOD at Tyr34; angiotensin-II induced hypertension in ratsIdentification of specific nitration at Tyr34; distribution of nitrated SOD in rat aorta; age-increase in nitrated SOD; distribution of nitrated SOD at Tyr34 in the kidney mainly in cortical collecting ducts[69,70]
MM-CKIP of CK from heart failure in rats; WB and immunohistochemistry; human biopsy of atrial fibrillationNitration and inactivation of MM-CK in heart; in vitro inactivation of MM-CK by ONOO but not myosin ATPase; selective reduction of MM-CK activity in human biopsies; increase nitration and carbonyl formation of MM-CK in atrial fibrillation[80,82]
α-actininIP of α-actinin from human cardiomyocytes and WB against 3-NO2-TyrContractile dysfunction of cardiomyocytes after exposure to ONOO; detection of nitrated α-actinin as the only nitrated protein at the concentration used[83]
SERCAIP of nitrated proteins from SR of age rat skeletal muscle; WB; protease digestion HPLC-ESI-MS; amino acid analysis; IP of SERCA 2a and Western Blot against 3-NO2-TyrNitration of Tyr294,295 in the M4–M8 transmembrane domain of SERCA 2a; correlation between reduction in Ca ATPase activity and nitration; age-dependent increase in nitration of SERCA 2a; nitration of Tyr294,295 in SERCA 2a in rat heart; nitration of SERCA 2a in patients with idiopathic dilated cardiomyopathy[86,88,89]
  • IP = immunoprecipitation; WB = Western blot; HPLC = high performance liquid chromatography; ESI = electro spray ionization; MS = mass spectrometry; GC = gas chromatography; FT-ICR = Fourier transformed-ion cyclotron resonance; Apo = apolipoprotein; COX = cyclooxygenase; PGIS = prostacyclin synthase; SOD = superoxide dismutase; MM-CK = myofibrillar creatine kinase; SERCA = sarcoplasmic reticulum Ca2+-ATPase; MPO = myeloperoxidase; CAD = coronary artery disease; LDL = low density lipoprotein; ONOO = peroxynitrite; Tyr = tyrosine; Tyr = tyrosyl radical; 3-NO2-Tyr = 3-nitrotyrosine and 3-Cl-Tyr = 3-chlorotyrosine.

6 I. Nitration of plasma proteins

Identification of circulating nitrated proteins is of great relevance in order to establish the relationship between nitroxidative stress and several pathological conditions in humans, especially in large clinical trials where readily accessible sampling is preferred. Although free and protein 3-NO2-Tyr has been detected in human plasma, data on the identity of specific proteins is scarce. Intravascular oxidatively-modified proteins have a high turnover and therefore a low steady state concentration. In fact, intravascular nitrated proteins are in equilibrium with other compartments (e.g. vascular wall [33]) where nitration occur, which makes difficult to asses the exact compartment where the protein was modified.

7 Fibrinogen

Fibrinogen is a soluble plasma protein that plays a major role during clot formation. Recent large clinical prospective trials point towards fibrinogen as being an independent risk factor for coronary artery disease and stroke [34]. In addition, clinical trials have shown an association between nitroxidative stress, and fibrinogen in patients with CAD [35,36]. A 30% increase in nitrated fibrinogen was detected in plasma of patients with documented CAD. The authors showed that levels of immunoprecipitated fibrinogen from plasma of patients with CAD reached 35 vs. 25.4 μmol/mol of tyrosine, of age-matched control subjects. This work expands on the functional consequences of nitrated fibrinogen showing that either nitrated fibrinogen from patients or in vitro modified fibrinogen polymerizes faster than normal fibrinogen. There are also changes in the architecture (increased in fibre ends and large pores) that led the authors to propose that modified molecules may cap the growing fibre thus only few modified molecules could have an amplifying effect on clot formation. Alterations of viscoelastic properties of clots arising from nitrated fibrinogen are compatible with less stable clots and therefore more susceptible to disruption by mechanical stress increasing the potential risk of microemboli. Plasmin is the effector enzyme of the fibrinolytic system that readily hydrolyzes the fibrin fibres during clot resolution. There are no differences in the proteloytic rate by plasmin or in the ability to aggregate and bind platelets between normal and nitrated fibrinogen [37]. However, plasmin has been shown to be nitrated in plasma of smokers [38]. Peroxynitrite inhibits and nitrates humans plasmin in vitro although rather high concentrations are needed (IC50=280 μM; plasmin 10 μM). The conversion of plasminogen to plasmin is also impaired by nitration [39].

8 Circulating nitrated lipoproteins

Pioneer work by Beckman et al. in the mid 1990s showed that nitrated proteins and cells are found in the atheroma plaques of patients with CAD [40] raising the possibility that nitrated lipoproteins were located on it. In the past few years, HDL dysfunction emerged as a new anomaly linked to nitroxidative stress in cardiovascular pathology [41]. Myeloperoxidase-mediated chlorination and nitration at tyrosine residues of apo A-I (major apoprotein of HDL) have been associated with an alteration in the cholesterol reverse transport by interfering with the ABCA-1 receptor [42]. Recently published data has shown that chlorination of Tyr192 is largely responsible for the impaired reverse cholesterol transport whereas nitration of the same residue has minimal effects [43]. It has been shown that human apo A-1 binds MPO thus facilitating site-specific tyrosine modification [44]. Nitrated circulating HDL in plasma of patients with CAD is two fold higher than control patients (136 vs. 68 μmol 3-NO2-Tyr/mol Tyr) [45]. Moreover, HDL appears to be a preferential target among circulating proteins for nitration since HDL 3-NO2-Tyr residues are 7–70 fold enriched in comparison with whole plasma protein 3-NO2-Tyr concentration [44,45]. These findings together with the fact that circulating levels of MPO are associated with an increased risk of CAD [46] suggest a connection between nitration of apo A-1, MPO and the underlying molecular mechanisms that operate in CAD.3

9 II. Nitration in the vessel wall

The artery wall is a major site of nitroxidative protein modification in several pathologies such as hypertension and atheromatosis. Despite its apparent simple anatomic organization (unicellular layer of endothelial cell, smooth muscle layer and adventitia), complex metabolic changes are held in the inflammatory micro environment reached during the inflammatory processes. It has been well documented that cytokine-activated endothelial cells, smooth muscle cells and fibroblast located in the atheroma lesion produce O2 [48]. Moreover, activated macrophages and neutrophils release large amounts of O2, NO and MPO at the injury site. Thus, several mechanisms of nitration are expected to occur in this compartment. Nitration reactions are favoured in the artery wall given that some of the detoxifying reactions that occur at the lumen (isomerization of ONOO by hemoglobin and direct reactions with sulphydryls) are less relevant for example in the extracellular matrix due to their low concentration. Each of the individual major risk factors for atheromatosis (hyperglycaemia, hypercholesterolemia, smoking and essential hypertension) produces endothelial dysfunction and therefore facilitates nitroxidative stress [49]. Atheromatosis is currently viewed as an inflammatory condition of the artery wall that develops over the years where modified pro-atherogenic LDL allocated at the neointima, triggers the local inflammatory response with leucocyte infiltration to the artery wall that perpetuates the process.

10 Nitration of LDL

Ample data exist on the oxidative mechanisms that lead to LDL modification and nitration. Peroxynitrite as well as MPO–H2O2–NO2 efficiently nitrates and starts lipid peroxidation processes in LDL in vitro [50].

Nitrated LDL triggers the release of TNF-α from non differentiated human monocytes, consequently amplifying the inflammatory process in vivo [51]. Oxidized, but mainly nitrated LDL [52] is taken up by macrophages via the unregulated scavenger receptor pathway (mainly SR-A and CD 36) leading to macrophage derived-foam cell formation [53].

Nitrated LDL from human thoracic aorta atheroma plaques [54] has been detected. Importantly the authors report a 90-fold increase in nitrated LDL obtained from the lesion than from matched plasma (840 vs. 9 mmol/mol of tyrosine) which strongly suggest that LDL is modified and retained at the artery wall within the atheroma plaque.

11 Nitration of cyclooxygenase and prostacyclin synthase

Eicosanoids are important players in the normal homeostasis of the endothelium. In platelets, cyclooxygenase 1 (COX-1) transforms arachidonic acid (AA) to thromboxane-A2 (TxA2) which is a potent agonist of platelet aggregation, thrombus formation and vasoconstrictor [55]. In a similar pathway, but on endothelial cells, arachidonic acid leads to the formation of prostacyclin (PGI2) through the actions of cyclooxygenase 2 (COX-2) and prostacyclin synthase (PGIS). Prostacyclin leads to an increase in cyclic AMP (AMPc) [56] that deactivates platelets, inhibits clot formation and is a potent vasodilator. As can be envisioned, NO and PGI2 act synergistically and as a reciprocal back up system to assure normal endothelium homeostasis [57]. Atheromatosis disrupts the physiological balance of autacoids, shifting it towards TxA2 and ONOO and therefore a pro thrombotic state. There is a complex interplay between NO-derived oxidants and the arachidonic acid pathway, which is beyond the scope of this review. In short, some intermediates derived from the NO pathway can either activate or inactivate cyclooxygenase depending on their nature and concentration [55,57,58]. For example, low levels of ONOO and other peroxides in presence of arachidonic acid, can activate COX but higher pathophysiological levels inhibit and nitrate COX [59]. Notably, COX activity requires the participation of a Tyr allocated at position 385, which is essential for activity as demonstrated by site-directed mutagenesis experiments [60]. Additionally, a Tyr at position 504, which participates in the catalytic cycle but is not essential for activity, has been identified by EPR studies [61]. It is well documented that ONOO but not NO2 or NO3 nitrates COX in vitro or cellular models at Tyr385 [62]. Furthermore, nitration is associated with inhibition of enzyme activity and has been detected in human atheroma plaques but not in control sample mammary artery [63]. Also, experiments with double knockout mice for Apo E receptor and iNOS support NO-derived nitration of COX in atheromatosis [64].

In endothelial and smooth muscle cells, COX 2 fuels PGIS for the generation of PGI2. Peroxynitrite readily nitrates and inactivates PGIS at extremely low concentration with a reported IC50≈100 nM [65]. Prostacyclin synthase depends on heme-thiolate prosthetic group for activity. The mechanistic aspects of ONOO reaction with heme-thiolate are similar to the previously described for heme-peroxidases. From a kinetic stand-point, heme-thiolate reacts faster than non-thiolate heme explaining in part the high sensitivity towards ONOO [66]. Recent studies have successfully identified the nitration site at Tyr430 with highly specific and sensitive mass spectrometry techniques [67].

The nitroxidative stress that takes place in atheromatosis affects profoundly the artery wall leading not only to a decrease of NO bioavailability but also to a decline of PGI2 levels due to nitration and inactivation of sensitive enzymes.

12 Mn-SOD

Mn-SOD is located in the mitochondrial matrix and is considered to be a fundamental detoxifying enzyme of these organelles since it is closely located to the respiratory chain, a major site of O2 production. Mn-SOD was one of the first nitrated proteins to be identified in chronic renal allograft rejection disease in humans [68]. Further studies have demonstrated that Mn-SOD is readily inhibited and nitrated at Tyr34 after reaction with ONOO [69]. Recently, nitrated Mn-SOD was identified in vascular aging on rats. Analysis by immunoelectron microscopy of tissue sections from aged aorta stained with anti 3-NO2-Tyr antibody revealed that nitrated proteins are located mainly at the mitochondria independently of the layer of the vessel wall studied. Moreover, although the authors found no differences in Mn-SOD levels with age, its activity diminishes, and in parallel, nitration of Mn-SOD increases [70] which strongly suggests a connection between nitration and inactivation in vivo. Finally, Mn-SOD nitration in endothelial cells after addition of Cyclosporine A (a potent immunosuppressant with known nephrotoxic and vascular side effects) due to peroxynitrite formation, has just been reported [71].

13 III. Nitration of the myocardium

The molecular basis of heart failure is poorly understood. A series of pathological changes at the molecular levels have been implicated. Among them, nitroxidative stress generated by inflammatory changes that occur after myocardial infarction, infectious process of the myocardium such as myocarditis, or other experimental set ups, have been linked to the alteration of energetic balance and contractile dysfunction of the failing heart [72,73].

14 Myofibrillar creatine kinase

Creatine kinase (CK) forms phosphocreatine (pCr) which serves as an energy reserve source for the heart. Myofibrillar CK exists either as a homodimer (MM-CK) or as a heterodimer (MB-CK). Both isoforms are expressed in skeletal and cardiac muscles. Although MB-CK is mainly found in myocardium (5–30% of total CK), MM-CK is responsible for the largest proportion of CK activity in this tissue. It is now well documented that there is a compromise on pCr pools (60% depletion) in animal models of heart failure [74]. This observation is extensive to humans, where recently proton nuclear magnetic resonance studies (H NMR) have demonstrated a depletion of pCr pools and its correlation with the severity of heart failure [75]. Actually, pCr/ATP ratio is a better predictor of cardiovascular mortality than the heart failure classification from the New York Heart Association, which underscores the importance of energetic balance in the failing heart [76]. Moreover, experiments with either acute [77] or chronic inhibition [78] of CK activity and experiments with knock out mice for CK have revealed a loss in the contractile reserve of the heart [79].

Pharmacological-induced [73] and post-ischemic [80] heart failure in animal models, are associated with an inactivation and nitration of MM-CK. The authors show that nitration of myocardium spread beyond the infarction scar and it is not associated with any cellular inflammatory infiltrate, which supports a ONOO mediated nitration mechanism. In addition, the IC50 of MM-CK for inactivation by ONOO is 93 μM while inactivation for mitochondrial CK starts at 200 μM (isolated mitochondria) [81]. This suggests that the myofibrillar structure is particularly sensitive to nitroxidative stress [80]. The same group successfully demonstrated that nitration but not other oxidative modifications of MM-CK correlates with enzyme inactivation in humans with atrial fibrillation undergoing surgery [82].

15 α-actinin

Another myofibrillar protein that is susceptible of nitration is α-actinin. Recent studies have shown that low doses of ONOO (IC50=55 μM) nitrates α-actinin in human isolated cardiomyocytes. The nitration is associated with contractile dysfunction as measured by maximal Ca2+-activated isometric force generated by ONOO-treated cardiomyocytes [83].

16 Sarcoplasmic reticulum Ca2+-ATPase

Calcium metabolism is under strict control throughout the muscle contraction–relaxation cycle. In the relaxation phase, cytosolic Ca2+ is pumped back to sarcoplasmic reticulum by SERCA. Alterations in Ca2+ homeostasis have been recognized for a long time in heart failure. Recent studies with transgenic mice confirm that cardiac contractility is modulated by SERCA levels [84]. There are several tissue-specific isoforms of SERCA with a disparity in its sensitivity to oxidative modifications. Among them SERCA-2a, found predominantly in slow twitch skeletal muscle, vessel smooth muscle and cardiac muscle, is sensitive to inhibition and nitration by ONOO. Early studies on SR vesicles exposed to rather high ONOO concentration (0.45 mM), showed inactivation of SERCA by sulphydryl oxidation that could be reverted by disulphide-reducing agents. [85]. More recently, the authors were able to show selective in vivo nitration of Tyr294 and Tyr295 of SERCA-2a from skeletal muscle during aging using a mass spectrometry approach [86]. In the same experimental conditions and in spite of the high sequence homology (≈84%), SERCA-1 was not significantly nitrated by authentic ONOO suggesting a unique sensitivity of SERCA-2a for nitration accompanied by inhibition of the ATPase activity.

Hypercholesterolemic rabbits show extensive nitration at the atheroma plaque that is reverted after butyl hydroxyl toluene (BHT) treatment [87]. In addition, nitrated SERCA was identified in the aorta smooth muscle layer, as evaluated by confocal microscopy. Importantly, immunoprecipitation of SERCA from human coronary artery atheroma was found to be nitrated and estimated to be 1.5 mol of 3-NO2-Tyr/mol protein, which is in agreement to previous finding reported for aged rats [87]. In human subjects with idiopathic dilated cardiomyopathy but normal coronaries, SERCA is nitrated. Isolation of cardiomyocytes from these patients also shows a significant correlation between time to half relaxation and 3-NO2-Tyr/SERCA ratio, which suggests that nitration can contribute to the cardiac dysfunction in heart failure [88]. Recent studies have been devoted to unravel the connection between nitroxidative modification of SERCA-2a and functionality in the senescent heart. The bis-nitration of two consecutive tyrosine residues at position 294 and 295 in the membrane-spanning helix-M4 of SERCA was confirmed [89]. These nitrated residues are located at the lumen site in a loop segment near a negatively charged glutamate residue. Finally, nitration of critical Tyr122 was identified in vitro, although it has not been detected from isolated SR vesicles. While the reason for this discrepancy is not solved, some authors have proposed an increased degradation of nitrated SERCA by the proteosome or the reduction of nitroTyr122 by accessory proteins (such as a putative denitrase activity) [90–92].

17 Hypertension and nitroxidative stress

Hypertension is associated with endothelial dysfunction and altered NO bioavailability in part by an increase in O2 production. Recently, a decrease in EC-SOD activity in hypertensive African Americans has been reported. This reduction in activity is not due to a decreased expression of EC-SOD and is associated with nitroxidative stress which was assessed by an augmentation in plasma protein nitration and plasma 8-isoptrostanes [93]. The identity of nitrated proteins in hypertension remains to be determined. Most importantly, people (heterozygotes) that posses a frequent common gene variant (allelic frequency=4%) with a mutation on the heparin binding domain of EC-SOD [94], are genetically predisposed to an increased risk for CAD [95].

18 In vivo determination of protein tyrosine nitration: issues on quantitative analysis

In vivo accurate detection of 3-NO2-Tyr represents an experimental challenge that deserves some consideration. The first approach utilized for the biochemical characterization of 3-NO2-Tyr, relayed in its spectrophotometrical properties which limit its detection to 10 μM, far beyond the reported basal value for free 3-NO2-Tyr in human plasma (1.5±1.0 nM). Several HPLC methods have been developed for the determination of 3-NO2-Tyr, which undoubtedly improve selectively and sensitivity, allowing to separate different analytes with similar characteristics, crucial for complex biological mixtures [20]. At present, the gold standard technique for the unambiguously detection of 3-NO2-Tyr is tandem mass spectrometry (MS/MS) coupled to either GC or HPLC (for review see [96,97]). In order to use free circulating 3-NO2-Tyr as an indicator of risk in the disease state, a normal basal level is mandatory. Although there is no general agreement in the literature and more studies are warranted, the normal range of circulating 3-NO2-Tyr is likely to be 1 nM. Hence, the limit of quantitation (LOQ) for the determination of 3-NO2-Tyr should be in 0.5 nM [96,97]. Circulating 3-NO2-Tyr possibly reflects the turnover of nitrated proteins since the modified amino acid cannot be reincorporated to de novo protein synthesis. Quantitation of protein 3-NO2-Tyr is another strategy to evaluate nitroxidative stress. The protein fraction of the biological sample is completely hydrolyzed to its amino acids before quantitation of 3-NO2-Tyr and Tyr. Results are typically expressed as moles of 3-NO2-Tyr /Tyr. Protein acid hydrolysis with HCl is effective in releasing Tyr and 3-NO2-Tyr and it has been successfully used while alkaline hydrolysis has been minimally explored [97]. The major artifact in the determination of either free or bound 3-NO2-Tyr is the acid-catalyzed nitration of tyrosine. The presence of nitrite in the biological samples and the acid precipitation of proteins or acid hydrolysis could yield artifactual nitration [98]. In this regard, careful sample handling and LC-MS/MS isotope dilution methodologies using labelled tyrosine and 3-NO2-Tyr represent major advances for quantitation purposes and control for artifactual nitration [29,99]. Although important discrepancies are reported in the literature, basal values for protein 3-NO2-Tyr are 0.4–1.6 μmol 3-NO2-Tyr/mol Tyr. Another widespread semi-quantitative strategy for the analysis of 3-NO2-Tyr, are assays based on either monoclonal or polyclonal anti 3-NO2-Tyr antibody such as ELISA, immunoblotting, epi fluorescence or confocal microscopy, immuno-electron microscopy [27] and flow cytometry [20]. It is important to note that immunocytochemistry and immunohistochemistry are more sensitive than Western blotting [100]. Both strategies can be combined to synergistically increase sensitivity and precise evaluation of a specific target protein. Immunoprecipitation in combination with protein hydrolysis for quantitation of nitrotyrosine or trypsin digestion for mass analysis of peptides containing 3-NO2-Tyr has yielded outstanding results and is the most common approach utilized by works reviewed in this article. Recently specific antibodies for nitrated proteins have been developed and tested [100]. Antibodies against nitrated SERCA at Tyr294,295 and nitrated Mn-SOD at Tyr94 have been successfully tested in angiotensin-II induced hypertension in rats and, most importantly, in human aortic and atrial myocardium biopsies from diabetic patients, where both Mn-SOD and SERCA stained positive.

19 Nitrotyrosine: a mediator or biomarker of cardiovascular disease?

Protein nitration is a usual process in the living organism and 3-NO2-Tyr accumulates during the aging process [32,70,89,100] reflecting the basal nitroxidative stress normally produced. Indeed, it is now well established that low levels of oxidants such as O2, H2O2 and presumably ONOO act at several signalling pathways (e.g. NF-kappa B, HIF-1 and angiotensin-II receptor) and are necessary for normal cellular function.

Nevertheless, as the physiological redox balance is weakened during the disease state, nitroxidative stress emerges as a mediator of damage, which includes among others, tyrosine nitration. The presence of nitrated proteins has been established in a myriad of cardiovascular diseases and is considered to be a marker of the aforementioned oxidant disbalance. Recent clinical trials have undoubtedly shown that plasma levels of 3-NO2-Tyr are correlated with the severity of CAD and that 3-NO2-Tyr levels diminish after statin treatment which strongly suggest a dependence on nitroxidative stress since statins are well known indirect antioxidants [101]. Although larger clinical trials are needed to confirm this observation, 3-NO2-Tyr is emerging as a consolidated marker of cardiovascular diseases. In addition, it can be used as a surrogate marker of nitroxidative stress in human trials to monitor the hypothesized beneficial action of either natural or synthetic antioxidants, which has been a regular absent variable in large and especially multi-centre clinical trials.

It is somewhat harder to establish if nitration of specific proteins is responsible for the molecular anomalies of a given disease or is an epiphenomenon. Some data discussed along this review clearly supports the former (nitration of PGIS).

Mechanisms that can explain the link between nitration and inactivation of enzyme activity include conformational changes, physico-chemical and/or steric restrictions at the active site. Protein tyrosine nitration is typically a low yield process (usually a few percentage of molecules of an individual protein are nitrated); hence, its impact in enzyme inhibition is expected to be low since the remaining unmodified enzyme molecules will be sufficient to accomplish the reaction. Another scenario of potentially greater biological significance is the gain-of-function after tyrosine nitration (e.g. nitrated fibrinogen that is prone to clot formation). In this case, a new activity arises that can amplify the biological signal in a cascade-like manner.

Finally, the mechanism of tyrosine nitration (Fig. 1) can help to rationalize pharmacological strategies to prevent tyrosine nitration and therefore recover the cardiovascular lesion, which is the proof-of-principle of the nitrated protein-mediated damage hypothesis. In this line, SOD-mimics and peroxynitrite-decomposition catalysts, e.g. manganese phorphyrins, (for review see [102]) have shown to decrease vascular protein tyrosine nitration and revert organ/tissue dysfunction in models of sepsis [103], heart senescence [104], angiotensin II-induced cerebral flow dysfunction [105] and post ischemic heart apoptosis [106]. In addition, novel approaches to inhibit protein tyrosine nitration and subsequent toxicity by the use of cell permeable tyrosine-containing peptides [107], should be tested in the context of cardiovascular pathology.


This work was supported by grants from the Howard Hughes Medical Institute and the International Centre of Genetic Engineering and Biotechnology and donation for research support by Laboratorios Gramón Bagó-Uruguay to R.R. G.P. was partially supported by a scholarship from Programa para la Investigación Biomédica (Pro. In. Bio.), Facultad de Medicina. We thank B.S. Valeria Valez for her assistance in artwork.


  • 1 The covalent substitution of a nitro (–NO2, +45 Da) moiety in either two of the ortho position carbon atoms (3-position) of the phenolic ring of a tyrosine residue gives rise to 3-nitrotyrosine. It is a posttranslational modification originated by nitric oxide-derived oxidants such as peroxynitrite and nitrogen dioxide (NO2).

  • 2 During inflammation, neutrophil degranulation releases MPO favouring this nitration pathway. The principal product of MPO is hypochlorous acid (HOCl) that can itself modify tyrosine to 3-chlorotyrosine with functional implications. Therefore, the simultaneous presence of 3-chlorotyrosine and 3-NO2-Tyr (MPO) or the concomitant formation of DOPA and 3-NO2-Tyr (ONOO) can aid in defining the nitration pathway implicated in a given situation (see also [1,5]).

  • 3 Albumin, the most abundant plasma protein, is oxidized at its single free Cys residue (Cys34) and nitrated at several Tyr. Surprisingly, nitrated albumin is not a frequent finding in human pathology. Nitrated proteins are immunogenic and autoantibodies against 3-NO2-Tyr have been detected in acute lung injury [47] although data in cardiovascular pathology has not been reported yet.


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View Abstract