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Vol. 54, Issue 4, 619-634, December 2002
Department of Integrative Biology and Pharmacology and Institute of Molecular Medicine, University of Texas Medical School, Houston, Texas
Abstract
I. Introduction
II. Oxidative Pathways in Cardiovascular Disease
A. Reactive Nitrogen and Oxygen Species
B. Sources of ·NO
C. Sources of O
D. Consequences of Oxidative Events
III. Mechanisms of Protein Nitration in Vivo
A. ONOO-Dependent Tyrosine Nitration
1. Tyrosine Nitration in Hydrophobic Conditions.
2. Tyrosine Nitration in the Absence of Heme Peroxidase.
B. Heme Peroxidase-Dependent Tyrosine Nitration
C. Other Putative Mechanisms
D. Selectivity of Protein Nitration
IV. Protein Nitration under Physiological Conditions
A. Oxidative Modification of Proteins and Redox Regulation
B. Protein Nitration
C. Feedback Regulation
D. Rationale for "Denitrase"
V. Protein Nitration in Cardiovascular Disease
A. Cardiovascular Inflammation
B. Autoimmune Myocarditis
C. Heart Failure
D. Ischemia-Reperfusion Injury
E. Cardiac Allograft Rejection
F. Transplant Coronary Artery Disease
G. Hypertension
H. Atherosclerosis
I. Diabetes
J. Cigarette Smoking
K. Aging
VI. Therapeutic Implications
VII. Future Directions
Acknowledgments
References
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There is growing evidence that cardiovascular disease is associated with progressive changes in the production of free radicals and radical-derived reactive species. These intermediates react with all major cellular constituents and may serve several physiological and pathophysiological functions. The nitration of protein tyrosine residues has been used as a footprint for in vivo production of radical and nonradical reactive species. Tyrosine nitration may alter protein function and metabolism and therefore, provides for further dysfunctional changes. This review focuses on an appearance of tyrosine nitrated proteins in cardiovascular tissues under different settings of cardiovascular disease. Sources of reactive species, putative mechanisms of protein nitration in vivo, as well as protein nitration under normal physiological conditions, are also described. The goal of this review is to attract more attention to identification of specific proteins, which undergo tyrosine nitration and to study a correlation between their altered function and pathology. Understanding how protein nitration affects disease progression may offer a unique option for design of antioxidant therapy for the treatment of cardiovascular complications. At the same time, protein nitration can be a biological marker of efficiency of antioxidant therapy.
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I. Introduction |
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Protein tyrosine nitration is a well established
post-translational modification occurring in a number of diseases
(Greenacre and Ischiropoulos, 2001
). Tyrosine nitration may affect
protein structure and function. A gain of function, as well as no
effect on function have been reported for some nitrated proteins (Gole et al., 2000; Balafanova et al., 2002). However, the inhibition of
function is a more common consequence of protein nitration (Ischiropoulos, 1998; Greenacre and Ischiropoulos, 2001). It has also
been shown that nitration of a tyrosine residue may prevent the
subsequent phosphorylation of that residue (Gow et al., 1996; Kong et
al., 1996). Alternatively, nitration of tyrosine residues may simulate
phosphorylation (MacMillan-Crow et al., 2000; Mallozzi et al., 2001)
and results in the constitutively active proteins. Furthermore,
tyrosine nitration may change the rate of proteolytic degradation of
nitrated proteins and favor either its faster clearance or the
accumulation of nitrated proteins in cells. Cumulatively, this suggests
that protein nitration may be involved in a variety of functions,
possibly including disease initiation and progression.
Many recent studies in cardiovascular research have demonstrated that there is an accumulation of nitrated proteins in different settings of cardiovascular disease. In this review, the following topics will be outlined: i) sources and mechanisms of protein nitration in vivo, ii) protein nitration in the cardiovascular system under physiological and pathological conditions, and iii) therapeutic implication of protein nitration.
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II. Oxidative Pathways in Cardiovascular Disease |
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A. Reactive Nitrogen and Oxygen Species
Nitric oxide (·NO) and superoxide (O
), dinitrogen tetroxide
(N2O4), dinitrogen trioxide
(N2O3), peroxynitrite (ONOO), peroxynitrous acid (ONOOH), alkyl
peroxynitrites (ROONO), nitronium cation (NO
There is emerging evidence that increased RNS/ROS production make a
significant contribution to the progression of cardiovascular disease
(Patel et al., 2000b; Cuzzocrea et al., 2001; Wattanapitayakul and
Bauer, 2001; Droge, 2002), but the actual sources of these reactive
species and mechanisms involved may not be identical in different settings.
B. Sources of ·NO
·NO is produced from L-arginine by the enzyme
nitric-oxide synthase (NOS). There are three isoforms of NOS: neuronal
NOS (nNOS or NOS-I) originally identified in brain, inducible NOS (iNOS or NOS-II) originally identified in macrophages, and endothelial NOS
(eNOS or NOS-III) originally identified in endothelial cells. Constitutive nNOS and eNOS require calcium and calmodulin as cofactors and generate low amounts of ·NO. Constitutively expressed
mitochondrial NOS was recently reported (Ghafourifar et al., 2001). Its
activity is also regulated by calcium. The iNOS that is expressed in
macrophages, endothelial cells, fibroblasts, vascular smooth muscle
cells and cardiac myocytes in response to inflammatory cytokines, does
not require calcium and calmodulin as cofactors. Furthermore, it
generates substantially larger amounts of ·NO for long periods
of time (Moncada et al., 1991; Nathan, 1992; Cannon et al., 1998;
Zweier et al., 2001). The expression of iNOS is regulated both at the
level of transcription and at the level of iNOS mRNA stability.
Catalytic activity of iNOS is regulated by the availability of the
substrate, L-arginine, and of the cofactors, NADPH and
tetrahydrobiopterin. Induction of iNOS expression is complemented by
co-induction of cationic amino acid transporter proteins (increase the
intracellular L-arginine level) and GTP cyclohydrolase (key
enzyme of tetrahydrobiopterin synthesis).
Increased ·NO production via induction of iNOS has been
suggested as a major mechanism by which cytokines mediate cardiac
contractile dysfunction and development of cardiovascular disease.
Indeed, iNOS mRNA and iNOS protein expression were demonstrated in many different settings of cardiovascular disease (Schulz et al., 1995; Wildhirt et al., 1995; Cannon et al., 1998; Sawyer and Colucci, 1998;
Zweier et al., 2001).
C. Sources of O
The major sources of intracellular O
The uncoupling of mitochondrial electron transport is a classical
mechanism of oxidant production with the developing consensus that
O). Under hypoxic or ischemic
conditions, the lack of oxygen supply disrupts the mitochondrial
electron transport chain, resulting in many adverse events (Lemasters
et al., 1997). Reoxygenation or reperfusion causes a massive production
of ROS due to the resumption of oxygen supply to mitochondrial
respiration (Lesnefsky et al., 1997). In addition to being a major
source of ROS, mitochondria are also a target for their damaging
effects. The phenomenon is that oxidative stress can lead to
dysfunctional mitochondria, and dysfunctional mitochondria may
self-amplify damage by generating further free radicals (Zorov et al.,
2000).
Nonmitochondrial sources of O). The
neutrophil NADPH oxidase may generate millimolar quantities of
O). Vascular
NADH/NADPH oxidase is activated by angiotensin II and significantly
contributes to O;
Wattanapitayakul et al., 2000).
Xanthine oxidase, a metalloflavoprotein, is involved in the purine
degradation pathway and generates O). Chronic
hypoxia or increased inflammatory cytokines can enhance xanthine
oxidase activity and also cause its release into the plasma. It was
shown, that the elevated levels of circulating xanthine oxidase
participate in endothelial dysfunction (Houston et al., 1999).
nNOS exhibits oxidase activity in the case of insufficient substrate or
tetrahydrobiopterin supply (Heinzel et al., 1992; Pou et al., 1992).
Cofactor-deficient nNOS cannot catalyze the five-electron oxidation of
L-arginine to ·NO, but it can receive electrons from
NADPH and donate them for one electron reduction of oxygen to
O) and endothelial (Vasquez-Vivar et al., 1998) NOS
isoforms, demonstrating that enzymatic generation of O).
However, recent studies (Xu, 2000a,b) suggested that NOS coenzyme and
cofactors might cause O
D. Consequences of Oxidative Events
The net concentrations of ·NO at the tissue level may
predict its protective or toxic effects. Many lines of evidence suggest that modulation of ·NO concentration will determine whether or
not the roles played by RNS/ROS will be protective or detrimental to
the cardiovascular system (Cannon et al., 1998; Ronson et al., 1999;
McCarty, 2000; Patel et al., 2000a,b; Wattanapitayakul and Bauer, 2001;
Zweier et al., 2001). Availability of ·NO is determined by the
amounts produced and by the local chemical environment, which promotes
either protection of ·NO by antioxidants or depletion of
·NO by O
Many studies conducted have illustrated that increased RNS/ROS
production may be a unifying mechanism in cardiovascular disease progression. Adverse changes associated with RNS/ROS production have
been found, essentially, at all levels of the cardiovascular system:
including gene expression, signal transduction, energy metabolism,
antioxidant defense and cell death (reviewed by Wattanapitayakul and
Bauer, 2001). Molecular mechanisms of these oxidative events include
post-translational modifications of proteins. Protein nitration is a
prominent one, which attracts much attention (Ischiropoulos, 1998;
Nakazawa et al., 2000; Greenacre and Ischiropoulos, 2001). However, it
is not clear whether protein nitration and subsequent alteration of
protein function contributes to progression of cardiovascular disease
or simply reflects the presence of complications caused by oxidative stress.
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III. Mechanisms of Protein Nitration in Vivo |
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Most of our knowledge regarding protein nitration is derived from
in vitro experiments with albumin or free tyrosine. The physiological
relevance of these findings remains to be defined. Presumably, the
nitration pathways in vivo are not mutually exclusive and may operate
simultaneously. Given the complexity of biological systems, it is
likely that the nitrating species responsible for protein nitration
must be evaluated for every model of disease separately. Meanwhile, the
mechanism(s) of in vivo nitration remains an area of active
investigation and controversy (Beckman, 1996; Eiserich et al., 1996;
Goldstein et al., 2000; Pfeiffer et al., 2000, 2001a,b; Reiter et al.,
2000; Sawa et al., 2000; Zhang et al., 2001a). The most likely
in vivo mechanisms for protein nitration are summarized in Fig.
1 and described below.
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A. ONOO-Dependent Tyrosine Nitration
Formation of ONOO by the diffusion-limited
recombination of ·NO with O; Radi et al.,
2001). A second order rate constant of this reaction was independently determined as 4.3, 6.7, and 1.9 × 109
M1 s1 (Huie and
Padmaja, 1993; Goldstein and Czapski, 1995; Kissner et al., 1997). It
has been suggested that ONOO can also be formed
by the reaction of nitroxyl anion (NO) with
O2 (Hogg et al., 1996). The latter reaction
proceeds at a slower rate (5.7 × 107
M1 s1) than that of
·NO with O
production from ·NO could be catalyzed by cytochrome
c (Sharpe and Cooper, 1998), and concentrations of
O2 in vivo are many orders of magnitude higher
than concentrations of O
ONOO is a strong oxidant capable of modifying
most biological molecules and compounds, including such amino acids as
tyrosine, tryptophan, cysteine, and methionine (Radi et al., 1991;
Alvarez et al., 1996, 1999). The detailed chemistry of the
ONOO-catalyzed reactions is beyond the scope of
this review but can be found in other sources (Beckman and Koppenol,
1996; Koppenol, 1998; Squadrito and Pryor, 1998; Ducrocq et al., 1999;
Radi et al., 2001). Nitration of free and protein-bound tyrosine to
yield nitrotyrosine is a well established in vitro reaction of
ONOO. However, there has been a debate over the
physiological significance of these findings (Goldstein et al., 2000;
Pfeiffer et al., 2000, 2001a,b). The major concern is that
ONOO formation and
ONOO-catalyzed tyrosine nitration both require
specific conditions that rarely occur in complex biological systems.
For example, a requirement of precisely balanced rates of ·NO
and O formation
(Pfeiffer and Mayer, 1998; Goldstein et al., 2000) or high
concentrations of potentially ONOO-specific
scavengers in biological samples (Mayer et al., 1998). However, another
study did not confirm the strict requirement of equimolar fluxes of
·NO and O (Jourd'heuil et al., 2001).
In addition, direct reactions of ONOO with
CO2, transition metals, and superoxide dismutase
(SOD) have been found to catalyze the nitration of tyrosine residues
(Beckman et al., 1992; Ischiropoulos et al., 1992; Lymar et al., 1996). These reactions increase the rate of tyrosine nitration and may explain
the ability of ONOO to nitrate proteins in vivo
despite the presence of high concentrations of compounds, such as
reduced glutathione, cysteine, or ascorbate, which act to inhibit
radical formation and therefore prevent nitration.
A few more considerations could be helpful in discussing a role of
ONOO in in vivo protein nitration. They include
1) tyrosine nitration in hydrophobic conditions and 2) tyrosine
nitration in the absence of heme peroxidase.
1. Tyrosine Nitration in Hydrophobic Conditions.
In terms of
the chemistry of tyrosine nitration, it seems likely that the local
environment of the targeted tyrosine residue may play a key role in
determining the final outcome of the reaction. Although much is known
about the chemistry of tyrosine nitration in aqueous solution, detailed
investigations of the chemistry of tyrosine nitration in the
hydrophobic interior of membranes or hydrophobic regions of proteins
have only recently begun (Goss et al., 1999; Zhang et al.,
2001a). ·NO and other oxides of nitrogen are hydrophobic
gases. They have higher solubility in hydrophobic solvents. This
suggests that the concentration of RNS may be higher in a hydrophobic
milieu. Even if the intrinsic rate constant of the RNS-mediated
reaction within hydrophobic phase is the same as in the aqueous
cytosol, the reaction is accelerated overall because of the increased
reactant concentration and the lack of the hydrolysis reaction (Liu et al., 1998b). The ONOO can freely pass
through lipid membranes, making ONOO-mediated
reactions in hydrophobic environment, such as cell membranes, organelles, lipoproteins, and sites buried in the protein tertiary structure, also of extreme relevance (Marla et al., 1997; Denicola et
al., 1998; Boulos et al., 2000; Khairutdinov et al., 2000; Zhang et
al., 2001a).
; Liu et al., 1998a).
The remarkable stability of this radical (a half-life of several days
at 25°C) is explained by the unique hydrophobic environment, which
stabilizes the tyrosyl radical. The formation of the tyrosyl radical
was suggested as a key element of tyrosine nitration. The possibility
of stabilizing tyrosyl radical in the hydrophobic environment could be
one more difference, which distinguishes tyrosine nitration in the
hydrophobic environment from tyrosine nitration in the hydrophilic environment.
However, the argument that tyrosine nitration occurs in a hydrophobic
environment is weakened by the fact that tyrosine residues mainly
don't intend to be buried away from solvent. Although, there is not a
universal method for measuring the relative affinities of amino acid
residues for hydrophobic phases, the consensus of different approaches
put the tyrosine residue in the middle of the hydrophobicity scale
(Eisenberg, 1984). This limits the probability of the tyrosine residue
appearing in the hydrophobic environment.
2. Tyrosine Nitration in the Absence of Heme Peroxidase.
Both
major mechanisms of protein tyrosine nitration,
ONOO- and heme peroxidase-dependent, probably
overlap in vivo. However, specific conditions in vivo can favor one
over the other. For example, it seems likely that mitochondria have no
heme peroxidases. At the same time, the mitochondrial respiratory chain
is a major source of O; Ghafourifar et al., 2001), the
intramitochondrial formation of ONOO is
becoming apparent. Indeed, evidence for intramitochondrial ONOO formation was presented in recent
publications (Ghafourifar et al., 1999; Valdez et al., 2000). Tyrosine
nitration of mitochondrial proteins is also recognized (MacMillan-Crow
et al., 1996, 2001; Park et al., 1999; Aulak et al., 2001; Riobo et
al., 2001; Turko et al., 2001; Yamamoto et al., 2002).
). At the same time, there was
no histological evidence of neutrophil infiltration into cardiac
tissue. These two observations together favor
ONOO as a source of tyrosine nitrated proteins
in the doxorubicin-treated mice.
B. Heme Peroxidase-Dependent Tyrosine Nitration
Besides ONOO, it has become recognized
that other reactions, such as nitrite-dependent heme peroxidase
reactions also may give a rise to protein tyrosine nitration in vivo
(Van der Vliet et al., 1997; Eiserich et al., 1998; Van Dalen et al.,
2000; Pfeiffer et al., 2001b; Brennan et al., 2002). It has been shown
that heme peroxidase enzymes (myeloperoxidases, eosinophil peroxidases, horseradish peroxidases) in the presence of nitrite and
H2O2 can nitrate different
proteins in heart homogenates (Sampson et al., 1998) or different pure
proteins (Van der Vliet et al., 1997; Wu et al., 1999). This occurs
through simultaneous oxidation of nitrite and tyrosine to nitrogen
dioxide radical and tyrosyl radical, respectively. The subsequent
reaction of these two radicals yields nitrotyrosine. Tyrosine nitration
under these conditions was exclusively inhibited by catalase and azide
(an myeloperoxidase inhibitor) but not by SOD. This suggests that the
mechanism of tyrosine nitration is
ONOO-independent.
Protein tyrosine nitration could be achieved by the direct oxidation of
nitrite by H2O2, but this
reaction requires nonphysiological concentrations of
H2O2. Alternatively,
nitrate can be oxidized by myeloperoxidase-derived hypochlorous acid to
form nitryl chloride, which is capable of nitrating protein tyrosine
residues (Eiserich et al., 1996; Panasenko et al., 1997). However,
other studies (Sampson et al., 1998; Ohshima et al., 1999) did not
confirm physiological relevance of this reaction.
Nitrate balance studies consistently conclude that a greater amount of
nitrite is excreted than can be accounted for by ingestion. Therefore,
there are endogenous sources of nitrite production, namely ·NO
and the products of ·NO metabolism (Oldreive and Rice-Evans,
2001). For example, in the vascular system, ·NO is rapidly
oxidized to nitrate by reaction with oxyhemoglobin or methemoglobin
(Radi, 1996). The reaction of ONOO with a wide
variety of biomolecules results in the production of nitrite (Pryor and
Squadrito, 1995). Since O).
The discrimination between these two mechanisms of tyrosine nitration
could be mainly associated with infiltration of activated phagocytes,
which contain high levels of heme peroxidases. Activated phagocytes,
such as eosinophils and neutrophils or monocytes, play a central role
in host defense mechanisms. However, the reactive intermediates formed
by these cells also can harm normal tissue and contribute to
inflammatory injury. Myeloperoxidase and eosinophil peroxidase are the
most abundant proteins in the activated phagocytes, and the state of
phagocytic activation has been described as one of the early events in
cardiovascular disease (Zahler et al., 1999; Frangogiannis et al.,
2002). The infiltration of activated phagocytes during chronic settings
of cardiovascular disease is not well established (Wattanapitayakul and
Bauer, 2001). It is most likely that chronic settings favor the
ONOO-dependent mechanism of protein nitration
over the heme peroxidase-dependent.
C. Other Putative Mechanisms
Other mechanisms relevant to in vivo conditions have been
described (McBride et al., 1999; Zhang et al., 2000; Grzelak et al.,
2001; Kilinc et al., 2001; Ogino et al., 2001). These mechanisms of
protein nitration vary slightly from those described above and are
based on pseudoperoxidase activity of hemoproteins, such as copper/zinc
superoxide dismutase (Cu/Zn-SOD), catalase, hemoglobin, and myoglobin.
Perhaps, these reactions reflect the putative toxicity of hemoproteins
as the potent oxidants capable of generating RNS/ROS and promoting
oxidative damage.
Despite the obvious protective role of different SOD, the ability of
SOD to produce strong oxidants can be damaging to cells. Initially, it
was believed that H2O2 was
metabolized by Cu/Zn-SOD to form hydroxyl radicals, which serve as a
source of oxidative damage (Yim et al., 1993). Recent studies showed
that tyrosine nitration could play a part in this damage. In the
presence of bicarbonate (HCO
). HCO). It was proposed that in the presence of
nitrite/H2O2,
HCO). This causes generation of nitrogen dioxide and
carbonate anion radicals with subsequent oxidation and nitration of
tyrosine residues. These reactions may generate multiple tyrosine
derivatives, including nitrotyrosine. Another study showed that
Cu/Zn-SOD or Mn-SOD in the presence of
·NO/H2O2 caused
nitration of phenol and oxidation of dihydrorhodamine-1,2,3 to
rhodamine-1,2,3 (McBride et al., 1999). The latter was
interpreted as production of ONOO.
Collectively, these studies suggest an alternative mechanism, which may
have in vivo implications to protein nitration.
Catalase is a heme peroxidase ubiquitously expressed throughout
mammalian tissues that is involved in protecting cells from oxidative
stress. Catalase can catalyze in vitro nitration of free tyrosine or
tyrosine residues of bovine serum albumin in the presence of
azide/H2O2 (Ogino et al.,
2001). Oxidation of azide by the
catalase/H2O2 system can
generate azidyl radicals. Subsequent reaction of the azidyl radicals
with oxygen generates ·NO. The involvement of these products in
the catalyze-dependent tyrosine nitration as well as its physiological
relevance remains to be understood.
Hemoglobin, the main component of the erythrocyte, is a
ONOO scavenger of physiological relevance
(Minetti et al., 2000). However, hemoglobin exhibits different
enzymatic activities (Giardina et al., 1995), including the
pseudoperoxidase activity (Bao and Williamson, 1997; Alayash et al.,
2001). Incubation of human hemoglobin with
nitrite/H2O2 was found to
induce self-nitration and nitration of bovine serum albumin (Grzelak et
al., 2001). The hemoglobin-catalyzed nitration is not enhanced by
HCO).
Another hemoprotein capable of catalyzing nitrotyrosine formation is
the myoglobin (Kilinc et al., 2001). This reaction is nitrite/H2O2-dependent with
a pH optimum of approximately 6.0. Most likely, it may occur under
acidic pH and low oxygen tension produced during myocardial ischemia.
D. Selectivity of Protein Nitration
Apart from the mechanism of tyrosine nitration, the selectivity of
protein nitration is also a subject of interest. It has been shown that
the process of tyrosine nitration is residue-, protein-, and
tissue-specific: not all tyrosine residues of a protein are nitrated
and not all proteins are targets for nitration (Ischiropoulos, 1998;
Souza et al., 1999). Certain proteins can be preferentially targeted
for nitration. This selectivity may depend not only on the composition
and structure of a given target, but also on its intracellular
concentration, localization, and interaction with other molecules.
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IV. Protein Nitration under Physiological Conditions |
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A. Oxidative Modification of Proteins and Redox Regulation
RNS/ROS exist in biological cells and tissues at low
concentrations under normal physiological conditions and are involved in the redox regulation of many physiological functions (Droge, 2002).
The balance between their rates of production and their rates of
clearance determines their concentrations by various antioxidant
compounds and enzymes. Redox regulation requires that this balance be
changed, either by an increase in RNS/ROS production or a decrease in
the activity of the antioxidant system. There are several mechanisms
for reestablishing the original redox state after such a temporary
imbalance. Elevated RNS/ROS concentrations typically induce the
expression of genes whose products exhibit antioxidative activity.
Moreover, the rate of RNS/ROS synthesis is regulated by different
feedback mechanisms, for example by direct inhibition of NOS by
·NO (Abu-Soud et al., 1995). The expression of iNOS is also
regulated at the transcriptional and post-transcriptional level by
signaling pathways that involve redox-responsive agents such as the
transcriptional factor nuclear factor-
B or mitogen-activated protein
kinases (MacMicking et al., 1997).
Redox regulation under physiological conditions is often associated
with oxidative derivatization of proteins. For example, certain
signaling cascades involving protein tyrosine kinases can be enhanced
by oxidative inhibition of protein tyrosine phosphatases (Hardwick and
Sefton, 1995). All protein tyrosine phosphatases share a common
sequence motif with a catalytically essential cysteine residue in the
active center that can be inactivated by
H2O2 (Denu and Tanner,
1998). H2O2 can also
enhance the stimulation of the insulin receptor tyrosine kinase
activity by insulin (Schmid et al., 1999). This redox effect is
probably mediated by the oxidative derivatization of any of four
cysteine residues in the tyrosine kinase domain of this membrane receptor.
B. Protein Nitration
Modification of cysteine residues described above represents a
common mechanism of redox regulation. Nitration of tyrosine residues in
proteins may also be important in redox regulation under physiological
conditions. Nitration of tyrosine residues in proteins induces the
change of tyrosine into a negatively charged hydrophilic nitrotyrosine
moiety and causes a marked shift of the local
pKa of the hydroxyl group from 10.07 in tyrosine to 7.50 in nitrotyrosine. This is expected to change the
function of a protein. A gain of function as well as no effect on
function were reported for tyrosine nitrated proteins (Gole et al.,
2000; Balafanova et al., 2002); however, the inhibition of function is
a much more common consequence of protein tyrosine nitration (Ischiropoulos, 1998; Greenacre and Ischiropoulos, 2001). Nitration of
a tyrosine residue may also prevent further phosphorylation of that
residue (Gow et al., 1996; Kong et al., 1996). Alternatively, nitration
of tyrosine residues may simulate phosphorylation (MacMillan-Crow et
al., 2000; Mallozzi et al., 2001) and results in the constitutively active proteins. There is also evidence that tyrosine nitration may
mimic regulatory cyclic adenylylation of a specific tyrosine residue
(Berlett et al., 1996, 1998).
Protein tyrosine nitration has been detected in numerous tissues under
apparently normal physiological conditions (Greenacre and
Ischiropoulos, 2001). In the cardiovascular system, basal protein
nitration was found in all major types of cells, such as myocytes,
endothelial cells, fibroblasts, and vascular smooth muscle cells
(Davidge et al., 1998; Frustaci et al., 2000; Kajstura et al., 2001).
Basal protein nitration was also found in plasma (Khan et al., 1998;
Marfella et al., 2001). Some of these nitrated proteins were
identified. Myofibrillar creatine kinase (Mihm et al., 2001a),
prostacyclin synthase in coronary arteries (Zou et al., 1999), and
heart succinyl-CoA:3-oxoacid CoA-transferase (Turko et al., 2001) were
demonstrated to be nitrated under normal physiological conditions.
Several structural proteins, such as myosin heavy chain, 
-actinin,
and desmin were also found nitrated in control atrial myocytes (Mihm et
al., 2001b). These data are consistent with the emerging perspective
that low levels of tyrosine nitration may be a physiological regulator
of a signaling pathway.
C. Feedback Regulation
It is widely accepted that nitration of tyrosine residues in vivo
is derived from enzymatically produced ·NO. This implies that
tyrosine nitration is a critical component of ·NO biochemistry
and could function as a negative feedback modulator of ·NO
production. The recent study on the murine lung epithelial cells
(Robinson et al., 2001) demonstrated that ONOO
treatment causes accumulation of nitrotyrosine in iNOS and inhibits ·NO production. ONOO-dependent
inhibition of iNOS may be a mechanism of attenuating iNOS activity at
inflammatory sites in vivo. ONOO can also
inhibit the activity of xanthine oxidase and O). Down-regulation of xanthine oxidase activity may
serve as the feedback to limit further ONOO
formation. Presumably, many other proteins associated with the RNS/ROS
functions can be regulated by tyrosine nitration.
D. Rationale for "Denitrase"
Protein nitration occurs under normal physiological conditions and
affects the function of many proteins. To be a regulatory mechanism,
protein nitration requires reversibility. Indeed, putative denitrase
activity was demonstrated in several publications (Gow et al., 1996;
Kamisaki et al., 1998; Kuo et al., 1999, 2002). This activity was
monitored by the decreased intensity of nitrotyrosine immunoreactive
bands in Western blots and increased nitrate levels in reaction
mixtures. However, neither an enzyme catalyzing this reaction nor a
product of this reaction was identified. The reversibility of protein
tyrosine nitration remains to be elucidated. Meanwhile, the biological
rationale for this type of enzymatic activity is summarized below.
In the presence of NAD(P)H and a corresponding reductase the
nitrotyrosine could be enzymatically reduced to the nitro anion radical
(Krainev et al., 1998). The nitro anion radical is then oxidized by
molecular oxygen to yield O
Instead of repair, the proteolytic degradation of tyrosine nitrated
proteins may discharge nitrated tyrosine residues. Indeed, the
accelerated degradation of mildly oxidized proteins is a normal cellular function. However, extensively oxidized proteins are poor
substrates for proteases and may accumulate in cells (Davies, 2001).
Furthermore, nitration of tyrosine can change chymotrypsin-like proteolytic selectivity. For example, chymotrypsin was found to be
capable of cleavage next to nitrated tyrosine residues but at a
considerably slower rate than next to unmodified tyrosine residues
(Souza et al., 2000).
Proteolytic degradation of tyrosine nitrated proteins actually results
in the appearance of free nitrotyrosine. Free nitrotyrosine in vivo
could also be derived from direct nitration of free tyrosine. The
levels of free nitrotyrosine vary in different tissues, (Greenacre and
Ischiropoulos, 2001) but were detected everywhere. It was demonstrated
that systematic administration of free nitrotyrosine markedly
attenuates the subsequent hemodynamic responses to 1- and
-adrenoceptor agonists in anesthetized rats (Kooy and Lewis, 1996).
Inhibition of the hemodynamic action of angiotensin II by free
nitrotyrosine may be involved in the pathogenesis of inflammatory conditions, such as atherosclerosis, ischemia-reperfusion, and sepsis,
where tyrosine nitration is favored (Kooy and Lewis, 1996). It was also
shown that physiological concentrations of free nitrotyrosine can
induce vascular and endothelial dysfunction of rat thoracic aorta
segments in vitro (Mihm et al., 2000).
Review of possible adverse functions of protein-bound or free nitrotyrosine assumes an apparent need for repair of this modification and warrants further research on putative denitrases.
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V. Protein Nitration in Cardiovascular Disease |
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A. Cardiovascular Inflammation
The induction of iNOS in response to pro-inflammatory cytokines or
endotoxin (bacterial lipopolysaccharide) has been implicated in
cardiovascular dysfunction. The production of large amounts of
·NO during inflammatory challenge leads to the formation of
RNS/ROS capable of oxidizing many biological molecules including
protein tyrosine nitration. Human autopsy specimens obtained from
patients with a diagnosis of sepsis demonstrated intense nitrotyrosine immunoreactivity in the endocardium, myocardium, and coronary vascular
endothelium and smooth muscle (Kooy et al., 1997). Following endotoxin
or interleukin-1 treatment, tyrosine nitrated proteins were found in
myocardium (Oyama et al., 1998; Cheng et al., 1999), aorta (Szabo et
al., 1995), plasma (Kamisaki et al., 1997), and cultured cardiomyocytes
(Combes et al., 2001). Immunohistochemical studies showed a
co-induction of iNOS, cyclooxygenase and protein tyrosine nitration in
endocardial endothelium and coronary arteriole endothelium in rabbits
after endotoxin administration (Mebazaa et al., 2001). The treatment
with NOS inhibitors prevented tyrosine nitration. Cytokine-induced
myocardial dysfunction was also associated with overproduction of
O). These data indicate that changed
equilibrium between ·NO and O). Altered protein functions caused by tyrosine nitration could be a portion of this pathogenesis.
B. Autoimmune Myocarditis
Acute viral myocarditis is a potentially lethal disease in humans.
Autoimmune myocarditis, an experimental model for human postviral heart
disease, could be induced in laboratory animals by injection of cardiac
myosin. It was shown that autoimmune heart disease is accompanied by
iNOS expression and accumulation of tyrosine nitrated proteins in
inflammatory macrophages as well as in cardiomyocytes (Bachmaier et
al., 1997; Ishiyama et al., 1997; Shin et al., 1998). Focal myocarditis
was sufficient to induce nitrotyrosine formation throughout the whole
heart muscle (Bachmaier et al., 1997). Aminoguanidine, the iNOS
inhibitor, prevented myocardial destruction, inflammatory cell
infiltration and decreased immunostaining for tyrosine nitrated proteins.
C. Heart Failure
The failing heart displays a disruption of fundamental regulatory
processes. Among them is a balance between generation of ·NO and
ROS (Saavedra et al., 2002). Altered cross talk between ·NO and
oxidative stress may cause protein nitration. Indeed, extensive cardiac
protein nitration was demonstrated in multiple settings of cardiac
failure (Ferdinandy et al., 2000; Cesselli et al., 2001; Feng et al.,
2001; Mihm et al., 2001a). iNOS is expressed in the myocardium after
myocardial infarction and in heart failure. Studies on the iNOS(
/)
mutant and wild-type mice demonstrated that iNOS expression after
myocardial infarction causes myocardial dysfunction and results in
higher mortality in wild-type compared with iNOS(/) mutant mice. At
the same time, myocardial infarction significantly increased the levels
of myocardial and plasma nitrotyrosine (Feng et al., 2001).
Accumulation of tyrosine nitrated proteins was also coupled with
apoptotic cell death in the paced dog heart. Myocyte, endothelial cell,
and fibroblast apoptosis was detected before the impairment of cardiac
function became apparent. Cell death increased with the duration of
pacing and followed progressively increased nitrotyrosine formation
(Cesselli et al., 2001).
Statins, hydroxymethylglutaryl coenzyme A reductase inhibitors,
attenuate angiotensin II-induced cellular signaling. Cerivastatin improved left ventricular remodeling after myocardial infarction and
decreased the nitrotyrosine protein level in rats (Bauersachs et al.,
2001).
Not much is known about specific proteins, which undergo nitration and
which altered function may contribute to myocardial dysfunction. Mihm
et al. (2001a) demonstrated that myofibrillar creatine kinase is highly
sensitive to nitration in cardiac failure in vivo. The myofibrillar
isoform of creatine kinase is an important controller of myocyte
contractility. The myocyte contraction depends upon complex and tightly
regulated high-energy phosphate production and utilization. The
energetics of myocyte contraction are severely altered in myocardial
dysfunction. Increased oxidative stress has been implicated in the
pathology of multiple cardiac disease states. Nitration of critical
tyrosine residues in the active site of creatine kinase and impairment
of its catalytic activity could be a link between increased oxidative
stress and myocardial dysfunction (Mihm et al., 2001b, 2002).
D. Ischemia-Reperfusion Injury
Reperfusion of ischemic myocardium is the definitive treatment to
attenuate myocardial injury. Unfortunately, reperfusion itself causes
additional tissue damage mediated by several factors including
inflammatory response and consequently altered production of RNS/ROS
(Wang and Zweier, 1996; Liu et al., 1997; Yasmin et al., 1997; Zweier
et al., 2001). RNS/ROS generation can cause oxidative modifications of
proteins. This could be a critical factor in post-ischemic myocardial injury.
Although the molecular mechanisms of injury remain to be elucidated,
many studies showed that repetitive episodes of ischemia-reperfusion caused an increased formation of nitrotyrosine in cardiac tissue. Furthermore, various competitive inhibitors of the NOS enzyme have been
shown to reduce the level of cellular protein nitration and to reduce
reperfusion injury in various settings (Wang and Zweier, 1996; Liu et
al., 1997; Yasmin et al., 1997; Mori et al., 1998; Hayashi et al.,
2001; Zhang et al., 2001b; Zweier et al., 2001; Baker et al.,
2002). Preconditioning of isolated rat hearts before subsequent
ischemia-reperfusion also reduced formation of free nitrotyrosine
measured in the perfusate (Csonka et al., 2001). All these studies
support a role of protein tyrosine nitration in the genesis of
post-ischemic myocardial injury. However, little is known about
specific protein targets for nitration.
A recent publication (Zou and Bachschmid, 1999) implicates prostacyclin
synthase. Prostacyclin synthase, an enzyme with antithrombotic, antiproliferative, and dilatory functions in the normal vasculature, was found to be nitrated and inactivated in isolated bovine coronary arteries following hypoxia-reoxygenation. The administration of NOS
inhibitors or SOD prevented nitration and inactivation of enzyme and
abolished coronary vasospasm induced by hypoxia-reoxygenation (Zou and
Bachschmid, 1999). The current conclusion is that nitration and
inactivation of prostacyclin synthase results in accumulation of
unmetabolized prostaglandin H2, which causes the
observed vasospasm.
E. Cardiac Allograft Rejection
Cardiac transplantation is an effective therapy for end-stage
heart failure. However, cardiac allograft rejection remains a problem
and is the leading cause of death in cardiac transplant recipients
after the first year. It is broadly accepted that the immunological and
inflammatory reactions in the myocardium are the major component of the
pathological changes observed during cardiac allograft rejection, but
the molecular mechanisms, which ultimately cause rejection, are not
completely understood. There is a large body of evidence that the death
of cardiac myocytes is the hallmark of cardiac allograft rejection and
that ·NO produced by macrophages infiltrating the myocardium or
by the cardiac myocyte itself is potentially cytotoxic to heart muscle cells (Szabolcs et al., 1996; Cannon et al., 1998).
During cardiac allograft rejection, there is significant release of
cytokines as a part of the immune response to foreign antigens present
in the cells of transplanted heart. Cytokines cause expression of iNOS,
which generates large amounts of ·NO for long periods of time.
iNOS mRNA, iNOS enzyme activity, and immunostaining for iNOS protein
were increased in macrophages, endothelial cells, vascular smooth
muscle cells, and cardiac myocytes in rejected cardiac allografts
(Szabolcs et al., 1996, 1998; Sakurai et al., 1999; Akizuki et al.,
2000; Wildhirt et al., 2001). All these studies also demonstrated the
accumulation of tyrosine nitrated proteins, suggesting that tyrosine
nitration may play a role in the rejection process. Experiments
with iNOS inhibitors, O,
2001; Sakurai et al., 1999; Akizuki et al., 2000; Wildhirt et al.,
2001).
F. Transplant Coronary Artery Disease
Transplant coronary artery disease is a major cause of late
mortality after cardiac transplantation in humans. Studies on tissue
sections from patients with transplant coronary artery disease revealed
iNOS expression in neointimal macrophages and smooth muscle cells.
Normal coronary arteries had no evidence of iNOS expression. Similar to
the setting of acute and chronic cardiac allograft rejection, iNOS
expression in human arteries with transplant coronary artery disease
was associated with extensive nitration of protein tyrosines (Ravalli
et al., 1998). Studies on atherosclerotic lesions from patients with
transplant coronary artery disease revealed colocalization of two
enzymes involved in the inflammatory response, iNOS and
cyclooxygenase-2. Protein nitrotyrosine was found in the same
distribution as that of iNOS and was colocalized with cyclooxygenase-2
in macrophages (Baker et al., 1999). These findings indicate that
protein tyrosine nitration might be involved in the process leading to
the development of transplant coronary artery disease.
G. Hypertension
Various experimental models of hypertension, including genetic and
induced by angiotensin II or by aortic banding, have implied that this
pathophysiological state is associated with endothelial dysfunction, increased O). Abdominal aortic coarctation above the renal arteries leads
to severe hypertension proximal to the site of stenosis. Western blot
analysis with anti-nitrotyrosine antibody revealed marked increase in
nitrotyrosine abundance in the heart and the aorta segment proximal to
the stenotic site in aortic-banded rats (Barton et al., 2001). The
enhanced protein tyrosine nitration after the exposure to
ONOO also was found in aortas from hypertensive
rats compared with normotensive Wistar-Kyoto rats (Cabassi et al.,
2001).
Angiotensin II is a natural regulator of blood pressure and a well
recognized participant in many cardiovascular diseases (Stroth and
Unger, 1999). It was shown that increased gene expression of several
subunits of NADH/NADPH oxidase and subsequent generation of
oxygen-derived free radicals (particularly O), and this
may be an important component of angiotensin II-mediated cardiovascular
disease (Rajagopalan et al., 1996; Laursen et al., 1997). Although the
role of angiotensin II in cardiovascular disease is established, the
molecular mechanisms by which it participates have not been elucidated.
Recent studies have shown that oxidant stress response to angiotensin
II includes extensive tyrosine nitration of proteins in the vascular
endothelium (Wattanapitayakul et al., 2000; Wang et al., 2001). This
protein nitration correlates with the extent of endothelial dysfunction observed and is probably associated with increased production of
ONOO at the early stage of angiotensin II
action. Angiotensin II is a peptide, and it could be a target for
tyrosine nitration caused by ONOO. Studies on
in vitro tyrosine nitration of angiotensin II demonstrated that
nitration of the tyrosine residue totally inhibits vasoconstrictive properties of angiotensin II in vivo (Ducrocq et al., 1998).
Studies on rat model of lead-induced hypertension point to enhanced
ROS-mediated inactivation of ·NO with sequential increase of
abundance of tyrosine nitrated proteins in many tissues, including
heart (Vaziri et al., 1999). Concomitant administration of vitamin E
ameliorated hypertension and tissue levels of nitrotyrosine. The
beneficial effects of vitamin E support the role of increased RNS/ROS
activity in the pathogenesis of hypertension.
H. Atherosclerosis
The pro- and anti-atherogenic role of ·NO is broadly
reviewed (Patel et al., 2000a,b). One explanation for pro-atherogenic role is the modification of proteins and lipids caused by RNS/ROS derived from altered ·NO metabolism. A series of studies
demonstrated protein nitration in human atherosclerotic tissue (Beckman
et al., 1994; Buttery et al., 1996; Leeuwenburgh et al., 1997; Luoma et
al., 1998; Cromheeke et al., 1999; Depre et al., 1999; Hunter et al.,
1999). Protein tyrosine nitration was associated with iNOS expression
and detected in iNOS-positive macrophage-rich lesions at different
stages of atherosclerosis (Luoma et al., 1998; Cromheeke et al., 1999). Furthermore, iNOS and nitrotyrosine immunoreactivity were detected in
complex heterogeneous cellular plaques, in relatively acellular fibrous
plaques, and in myointimal plaques (Hunter et al., 1999). The presence
of iNOS and nitrotyrosine in plaque also correlated with plaque
instability in patients (Depre et al., 1999; Hunter et al., 1999).
Presumably, tyrosine nitrated proteins with altered function may
promote atherogenesis, counteracting the well established anti-atherogenic effects of ·NO.
Specific protein targets for nitration in atherosclerosis remain to be
identified. A recent study (Zou et al., 1999) on bovine atherosclerotic
arteries revealed tyrosine nitration of prostacyclin synthase. This
study focused on the early stages of atherosclerosis, when arteries
display focal thickening without signs of necrosis or rupture of
plaques. It is likely that earlier nitration and inactivation of
prostacyclin synthase and subsequent accumulation of pro-thrombotic
prostaglandin H2 may predispose further platelet aggregation and thrombus formation.
I. Diabetes
Diabetes causes early development of cardiovascular complications
(Grundy et al., 1999). There is emerging evidence that RNS/ROS make a
significant contribution to the progression of diabetes and its
complications (Honing et al., 1998; Rosen et al., 2001). Several recent
publications focused on diabetes-associated protein nitration (Frustaci
et al., 2000; Ceriello et al., 2001, 2002a,b; Kajstura et al., 2001;
Marfella et al., 2001; Turko et al., 2001). It was shown that the
apoptosis of myocytes, endothelial cells, and fibroblasts in heart
biopsies taken from diabetic patients is associated with intracellular
levels of nitrotyrosine (Frustaci et al., 2000). A positive correlation
between accumulation of nitrotyrosine and myocyte apoptosis in the
diabetic heart was also demonstrated (Kajstura et al., 2001).
Furthermore, perfusion of isolated rat hearts in conditions of high
glucose concentration was accompanied by the formation of nitrotyrosine
and evident cardiac cell apoptosis (Ceriello et al., 2002a). These
support the concept of oxidative stress as a mediator of the vascular
damage caused by hyperglycemia. These also consider protein tyrosine
nitration as a marker of oxidative damage in diabetes. Marfella et al.
(2001) showed that acute hyperglycemia in normal subjects causes an
oxidative stress as evidenced by the raised circulating protein
nitrotyrosine levels during the hyperglycemic clamp. Ceriello et al.
(2001) demonstrated that nitrotyrosine plasma levels were correlated
with plasma glucose concentrations in type II diabetic patients. They
also demonstrated that postprandial hyperglycemia is accompanied by
nitrotyrosine generation (Ceriello et al., 2002b). These observations
may have important implications for the pathogenesis of vascular
dysfunction in diabetes, if the pathway(s) for the increase of
protein-bound nitrotyrosine levels will be established.
Recently, we found that the mitochondrial protein
succinyl-CoA:3-oxoacid CoA transferase undergoes tyrosine nitration in
the rat heart following streptozotocin administration (Turko et al., 2001). To our knowledge, this is the first study to identify the increase of tyrosine nitration of a specific protein in diabetes. Succinyl-CoA:3-oxoacid CoA transferase is located in the mitochondrial matrix and catalyzes the formation of acetoacetyl-CoA from acetoacetate (Laffel, 1999). This is the rate-determining step of ketone body conversion into acetyl-CoA, which subsequently enters the citric acid
cycle. Diabetes is associated with a variety of abnormalities in
myocardial energy metabolism (Sato et al., 1995). Accumulating evidence
has implicated changes in myocardial energy substrate use as a
contributing factor to diabetes-associated cardiomyopathies (Stanley et
al., 1997). Our finding that succinyl-CoA:3-oxoacid CoA transferase
undergoes tyrosine nitration and exhibits lower catalytic activity in
the diabetic heart (Turko et al., 2001) is consistent with the
postulated shift in the source of acetyl-CoA for the citric acid cycle
in diabetic hearts (Stanley et al., 1997).
Endothelial dysfunction is a critical initial factor in the development
of diabetic vascular disease (Laight et al., 2000). Exposure of human
aortic endothelial cells to high glucose (30 or 44 mM) results in
tyrosine nitration and inactivation of prostacyclin synthase (Zou et
al., 2002). This can change thromboxane/prostaglandin H2 receptor stimulation and explain an increased
endothelial apoptosis in diabetes.
J. Cigarette Smoking
Cigarette smoking, as well as secondhand smoke, is considered a
risk factor for cardiovascular disease, but the mechanism of the
adverse effect of smoking is not fully understood. Cigarette smoke
contains abundant free radicals including ·NO. A shared feature
among cardiovascular disease risk factors is the generation of
increased RNS/ROS. Hence, cigarette smoke may induce some of its
damaging effects by free radical mechanisms. It was shown that exposure
to cigarette smoke extracts, prepared by bubbling the gas phase of
smoke into phosphate-buffered saline, converts free tyrosine to
nitrotyrosine (Yamaguchi et al., 2000). Exposure of plasma to gas-phase
cigarette smoke causes depletion of antioxidants, induces lipid
peroxidation, and is capable of converting tyrosine to nitrotyrosine in
proteins (Eiserich et al., 1995). Human plasma proteins, such as
fibrinogen, transferrin, plasminogen, and ceruloplasmin, were found to
have tyrosine nitrated residues in active smokers (Pignatelli et al.,
2001). Studies on the platelets from chronic smokers demonstrated
intra-platelet nitrotyrosine formation, which was associated with
increased platelet aggregation and with lower intra-platelet levels of
reduced glutathione and ascorbate (Takajo et al., 2001). Oral
administration of ascorbate to smokers restored these parameters
compared with the nonsmokers group. The data suggest that cigarette
smoke may cause damage of biomolecules, including tyrosine nitration of
proteins, and that endogenous antioxidants can attenuate some of these
adverse effects.
A recent study implicated tyrosine nitration of mitochondrial MnSOD in
hearts from mice exposed to cigarette smoke (Knight-Lozano et al.,
2002). Exposure to cigarette smoke also caused increased mitochondrial
DNA damage. These data together support the concept that
intramitochondrial RNS/ROS levels increase with cardiovascular disease
risk factor, cigarette smoking. Chronic exposure to cigarette smoke
could ultimately result in mitochondrial dysfunction, an important
early event in cardiovascular disease caused by oxidative stress
(Knight-Lozano et al., 2002).
K. Aging
Cardiovascular disease increases in frequency with age, even in
the absence of established risk factors. The underlying molecular mechanisms associated with age-related cardiovascular disease have not
been elucidated, but might involve impaired ·NO activity (McCann
et al., 1998). For example, the endothelium-dependent relaxation
declines with increasing age (Tschudi et al., 1996). Another recent
study (Van der Loo et al., 2000) demonstrated that there is an
accumulation of tyrosine nitrated proteins in the aortas of old rats
compared with aortas of young rats. One of the nitrated proteins
identified was Mn-SOD. Nitration of Mn-SOD leads to significant
reduction of its activity (Yamakura et al., 1998; MacMillan-Crow et
al., 1999). Mn-SOD is the major antioxidant enzyme in the mitochondria,
and even partial inhibition can have adverse consequences. It is also
likely, that the degree of Mn-SOD nitration may be a molecular marker
of vascular aging (Van der Loo et al., 2000).
One more example of age-related nitration of a specific protein is
reported for sarcoplasmic reticular Ca-ATPase isolated from the
skeletal muscle (Schoneich et al., 1999; Viner et al., 1999).
Inactivation of Ca-ATPase and decreased ATP utilization during aging
may represent an adaptive response that functions to down-regulate
energy metabolism and the associated generation of RNS/ROS (Squier,
2001).
The studies mentioned above indicate that protein nitration can have
both detrimental and protective effects in aging. However, another
study (Leeuwenburgh et al., 1998) indicated no significant increase in
levels of nitrotyrosine in heart, skeletal muscle, and liver from young
and old female rats.
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VI. Therapeutic Implications |
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TopPreviousNextReferences
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Major therapies for cardiovascular disease include drugs that
affect vascular tone, cardiac contractility, fluid status, or lipid
levels (Wattanapitayakul and Bauer, 2001). However, in light of the
critical roles of RNS/ROS in cardiovascular disease, it seems
reasonable to expect that antioxidant therapy may also have value.
Perspectives of antioxidant therapy for cardiovascular disease were
recently reviewed (Cuzzocrea et al., 2001; Snoeckx et al., 2001;
Wattanapitayakul and Bauer, 2001). They mainly include different
interventions to reduce RNS/ROS generation, such as antioxidant food
supplements, ONOO decomposition catalysts,
SOD-mimics, and modulations of expression of antioxidant enzymes,
including molecular chaperones.
The susceptibility to oxidative stress is a function of the overall
balance between factors that exert oxidative stress and those that
exhibit antioxidant capability. Oxidative damage can, therefore, be
described as a consequence of excessive oxidative stress and/or
insufficient antioxidant potential. Under normal physiological
conditions, ·NO, O, and
other RNS/ROS are part of the delicately balanced redox regulation.
Once it was altered by cardiovascular disease, the development of
procedures for reestablishing the original balance may be a central
issue of research on cardiovascular disease. However, flooding the
system with antioxidants or the overexpression of antioxidative enzymes
may be just as harmful as excessive exposure to RNS/ROS (Droge, 2002).
The major problem is the identification of the narrow line that
separates advantageous and detrimental effects of RNS/ROS. Protein
nitration is a type of oxidative damage that occurs in cardiovascular
disease. Many studies summarized in this review found a positive
correlation between protein nitration and cardiovascular disease
progression and concluded that protein nitration can be a biological
marker of disease progression. At the same time, protein nitration can
also be a biological marker of effectiveness of antioxidant therapy.
Such an example is brought up by studies on statin therapy. Statins,
cholesterol-lowering agents, can also improve the stability of the mRNA
for eNOS and enhance the generation of ·NO (Lefer et al., 2001).
It was demonstrated in an experimental model of myocardial infarction,
that cerivastatin treatment improved left ventricular remodeling,
whereas decreased the level of tyrosine nitrated proteins (Bauersachs
et al., 2001). Another study showed that simvastatin treatment reduced
the atherosclerotic area in the thoracic aorta of rabbits fed a 0.5%
cholesterol diet and, at the same time, decreased nitrotyrosine
staining (Thakur et al., 2001). It is recognized that eNOS-derived
·NO is a potent inhibitor of leukocyte recruitment at sites of inflammation. It seems likely that eNOS-derived ·NO does not
cause protein nitration, and protein nitration could still be a marker
of the efficiency of statin therapy.
Another question is whether altered functions of tyrosine nitrated
proteins contribute to disease development. Basically, the answer to
this question requires identification of a specific tyrosine nitrated
protein and a positive correlation between altered function of this
protein and development of complications. Several studies (Zou and
Bachschmid, 1999; Zou et al., 1999; Van der Loo et al., 2000;
Knight-Lozano et al., 2002; Mihm et al., 2001a, 2002) fit these
requirements and propose that protein nitration is a link between
increased oxidative stress and cardiovascular complications.
Collectively, this suggests that the search for antioxidants that
prevent protein nitration may offer a unique therapeutic option for the
treatment of cardiovascular complications.
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VII. Future Directions |
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TopPreviousNextReferences
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The emerging experimental data summarized in this review show protein nitration as an internal feature of cardiovascular disease (Fig. 2). The question is whether protein nitration occurs at an early stage of cardiovascular disease, contributing to the development of complications, or whether it is merely a consequence of the oxidative tissue damage, reflecting the presence of complications. The question is important and warrants future studies on detailed profiling of tyrosine nitrated proteins in different settings of cardiovascular disease. Additionally, function and metabolism of tyrosine nitrated proteins remain an area of research in need of rigorous study.
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Acknowledgments |
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This work was supported in part by grants from the National Institutes of Health (HL64221), the John S. Dunn Foundation, G. Harold and Leila Y. Mathers Charitable Foundation, The Welch Foundation, U.S. Army, and the University of Texas.
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Footnotes |
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Address correspondence to: Dr. Ferid Murad, Department of Integrative Biology and Pharmacology, University of Texas-Houston Medical School, 6431 Fannin, Houston, TX 77030. E-mail: Ferid.Murad{at}uth.tmc.edu
1 RNS, reactive nitrogen species; ROS, reactive oxygen species; NOS, nitric-oxide synthase; nNOS, neuronal NOS; iNOS, inducible NOS; eNOS, endothelial NOS; SOD, superoxide dismutase.
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/) and nitric synthase-2(+/+) mice: effects of cellular chimeras on myocardial inflammation and cardiomyocyte damage and apoptosis.
Circulation
103:
2514-2520
0031-6997/02/5404-619-634$7.00
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Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
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 B. Blanchard-Fillion, D. Prou, M. Polydoro, D. Spielberg, E. Tsika, Z. Wang, S. L. Hazen, M. Koval, S. Przedborski, and H. Ischiropoulos Metabolism of 3-nitrotyrosine induces apoptotic death in dopaminergic cells. J. Neurosci., June 7, 2006; 26(23): 6124 - 6130. [Abstract] [Full Text] [PDF]
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 S. Ghosh, A. J. Janocha, M. A. Aronica, S. Swaidani, S. A. A. Comhair, W. Xu, L. Zheng, S. Kaveti, M. Kinter, S. L. Hazen, et al. Nitrotyrosine Proteome Survey in Asthma Identifies Oxidative Mechanism of Catalase Inactivation J. Immunol., May 1, 2006; 176(9): 5587 - 5597. [Abstract] [Full Text] [PDF]
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 I. Dalle-Donne, R. Rossi, R. Colombo, D. Giustarini, and A. Milzani Biomarkers of Oxidative Damage in Human Disease Clin. Chem., April 1, 2006; 52(4): 601 - 623. [Abstract] [Full Text] [PDF]
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 H. Zhang, Y. Xu, J. Joseph, and B. Kalyanaraman Intramolecular Electron Transfer between Tyrosyl Radical and Cysteine Residue Inhibits Tyrosine Nitration and Induces Thiyl Radical Formation in Model Peptides Treated with Myeloperoxidase, H2O2, and NO2-: EPR SPIN TRAPPING STUDIES J. Biol. Chem., December 9, 2005; 280(49): 40684 - 40698. [Abstract] [Full Text] [PDF]
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 R. S. Baliga, A. A. Chaves, L. Jing, L. W. Ayers, and J. A. Bauer AIDS-related vasculopathy: evidence for oxidative and inflammatory pathways in murine and human AIDS Am J Physiol Heart Circ Physiol, October 1, 2005; 289(4): H1373 - H1380. [Abstract] [Full Text] [PDF]
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J. Kanski, S. J. Hong, and C. Schoneich Proteomic Analysis of Protein Nitration in Aging Skeletal Muscle and Identification of Nitrotyrosine-containing Sequences in Vivo by Nanoelectrospray Ionization Tandem Mass Spectrometry J. Biol. Chem., June 24, 2005; 280(25): 24261 - 24266. [Abstract] [Full Text] [PDF]
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W. Zhou, X.-L. Wang, T. L. Kaduce, A. A. Spector, and H.-C. Lee Impaired arachidonic acid-mediated dilation of small mesenteric arteries in Zucker diabetic fatty rats Am J Physiol Heart Circ Physiol, May 1, 2005; 288(5): H2210 - H2218. [Abstract] [Full Text] [PDF]
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P. Francia, C. delli Gatti, M. Bachschmid, I. Martin-Padura, C. Savoia, E. Migliaccio, P. G. Pelicci, M. Schiavoni, T. F. Luscher, M. Volpe, et al. Deletion of p66shc Gene Protects Against Age-Related Endothelial Dysfunction Circulation, November 2, 2004; 110(18): 2889 - 2895. [Abstract] [Full Text] [PDF]
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 A. Svatikova, R. Wolk, H. H. Wang, M. E. Otto, K. A. Bybee, R. J. Singh, and V. K. Somers Circulating free nitrotyrosine in obstructive sleep apnea Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2004; 287(2): R284 - R287. [Abstract] [Full Text] [PDF]
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T. Koeck, X. Fu, S. L. Hazen, J. W. Crabb, D. J. Stuehr, and K. S. Aulak Rapid and Selective Oxygen-regulated Protein Tyrosine Denitration and Nitration in Mitochondria J. Biol. Chem., June 25, 2004; 279(26): 27257 - 27262. [Abstract] [Full Text] [PDF]
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 T. Koeck, B. Levison, S. L. Hazen, J. W. Crabb, D. J. Stuehr, and K. S. Aulak Tyrosine Nitration Impairs Mammalian Aldolase A Activity Mol. Cell. Proteomics, June 1, 2004; 3(6): 548 - 557. [Abstract] [Full Text] [PDF]
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Y.-R. Chen, C.-L. Chen, W. Chen, J. L. Zweier, O. Augusto, R. Radi, and R. P. Mason Formation of Protein Tyrosine ortho-Semiquinone Radical and Nitrotyrosine from Cytochrome c-derived Tyrosyl Radical J. Biol. Chem., April 23, 2004; 279(17): 18054 - 18062. [Abstract] [Full Text] [PDF]
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S. FOGLI, P. NIERI, and M. C. BRESCHI The role of nitric oxide in anthracycline toxicity and prospects for pharmacologic prevention of cardiac damage FASEB J, April 1, 2004; 18(6): 664 - 675. [Abstract] [Full Text] [PDF]
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A. Martinez-Ruiz and S. Lamas S-nitrosylation: a potential new paradigm in signal transduction Cardiovasc Res, April 1, 2004; 62(1): 43 - 52. [Abstract] [Full Text] [PDF]
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 C. E. Berry and J. M. Hare Xanthine oxidoreductase and cardiovascular disease: molecular mechanisms and pathophysiological implications J. Physiol., March 15, 2004; 555(3): 589 - 606. [Abstract] [Full Text] [PDF]
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C. Vadseth, J. M. Souza, L. Thomson, A. Seagraves, C. Nagaswami, T. Scheiner, J. Torbet, G. Vilaire, J. S. Bennett, J.-C. Murciano, et al. Pro-thrombotic State Induced by Post-translational Modification of Fibrinogen by Reactive Nitrogen Species J. Biol. Chem., March 5, 2004; 279(10): 8820 - 8826. [Abstract] [Full Text] [PDF]
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A. A Chaves, M. J Mihm, B. L Schanbacher, A. Basuray, C. Liu, L. W Ayers, and J. Anthony Bauer Cardiomyopathy in a murine model of AIDS: evidence of reactive nitrogen species and corroboration in human HIV/AIDS cardiac tissues Cardiovasc Res, October 15, 2003; 60(1): 108 - 118. [Abstract] [Full Text] [PDF]
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I. V. Turko, L. Li, K. S. Aulak, D. J. Stuehr, J.-Y. Chang, and F. Murad Protein Tyrosine Nitration in the Mitochondria from Diabetic Mouse Heart: IMPLICATIONS TO DYSFUNCTIONAL MITOCHONDRIA IN DIABETES J. Biol. Chem., September 5, 2003; 278(36): 33972 - 33977. [Abstract] [Full Text] [PDF]
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D. M. Fries, E. Paxinou, M. Themistocleous, E. Swanberg, K. K. Griendling, D. Salvemini, J. W. Slot, H. F. G. Heijnen, S. L. Hazen, and H. Ischiropoulos Expression of Inducible Nitric-oxide Synthase and Intracellular Protein Tyrosine Nitration in Vascular Smooth Muscle Cells: ROLE OF REACTIVE OXYGEN SPECIES J. Biol. Chem., June 13, 2003; 278(25): 22901 - 22907. [Abstract] [Full Text] [PDF]
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