Cellular Information Processing
Mitochondria and regulated tyrosine nitration
T. Koeck1 , D.J. Stuehr and K.S. Aulak1
Department of Pathobiology, NC2, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, U.S.A.
Abstract
The conditions of the cellular microenvironment in complex multicellular organisms fluctuate, enforcing
permanent adaptation of cells at multiple regulatory levels. Covalent post-translational modifications of
proteins provide the short-term response tools for cellular adjustment and growing evidence supports the
possibility that protein tyrosine nitration is part of this cellular toolkit and not just a marker for oxidative
damage. We have demonstrated that protein tyrosine nitration fulfils the major criteria for signalling and
suggest that the normally highly regulated process may lead to disease upon excessive or inappropriate
nitration.
Cellular environmental adaptation
In complex multicellular organisms, cells are in a constant
need to adapt their phenotype to their function and microenvironment. These adaptations include responses to metabolic substrate availability, oxygenation status, osmolarity,
and mechanical forces like shear stress. Adaptation may be
performed by altering the concentrations and expression
profiles of proteins through gene transcription [1,2], translation/mRNA stability [1,3], as well as regulated protein
degradation [1,4], and/or by modulating protein function
by post-translational modifications [1,2]. While regulating
protein levels is important, the fine-tuning necessary for
sensing and responding to short-term microenvironmental
alterations requires post-translational mechanisms.
Cellular energy homoeostasis –
phosphorylation and nitric oxide (NO)
A critical process in living cells, depending on short-term
adjustments and tight regulation, is the maintenance of cellular energy balance, hence ATP flux. Matching ATP supply
and demand at the cellular level depends on the interaction
of metabolic pathways in cytosol and mitochondria with
oxygen level [5]. Protein phosphorylation provides crucial
reversible adjustments for sensing and regulating ATP flux.
It contributes to the activation of the AMP-dependent
protein kinase pathway [6] and regulates the activities of key
enzymes like pyruvate kinase [7,8], pyruvate dehydrogenase
[9] and 6-phosphofructo-2-kinase/fructose bisphosphatase 2
[10], thus controlling its interaction with glucokinase, altering
the compartmentation and activity [11,12]. Recently, tyrosine
phosphorylation was shown to modulate activity of the
MRC (mitochondrial respiratory chain) complex I and Fo F1 ATP synthase [13]. Thus phosphorylation takes part in adKey words: denitration, metabolism, mitochondrion, nitric oxide, oxidative stress, tyrosine
nitration.
Abbreviations used: ψ, electrochemical gradient; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; MnSOD, mitochondrial manganese superoxide dismutase; MRC, mitochondrial
respiratory chain; NOS, nitric oxide synthase; PI3K, phosphoinositide 3-kinase; RNS, reactive
nitrogen species.
1
Correspondence may be addressed to either of these authors (email [email protected] or
[email protected]).
justment of rate, yield and source of cellular ATP production
by modulating the activities of the oxygen-dependent mitochondrial oxidative phosphorylation as well as glycolysis
[14].
The highly diffusible free radical NO appears to be another
part of this metabolic adaptation by reversibly binding to
cytochrome c oxidase (complex IV) of the MRC in competition with oxygen [15]. This leads to inhibition of complex
IV and therefore ATP production through mitochondrial
oxidative phosphorylation at physiologically relevant NO
concentrations, with the degree of inhibition depending on
the local NO/O2 ratio [16]. As a consequence, an increasing
NO/O2 ratio, as is likely to occur during acute and chronic
mild-to-moderate hypoxia, results in decreased mitochondrial oxygen consumption, which delays severe hypoxia/
anoxia, and so supports the re-establishment of a regular
oxygen gradient and prevents excessive generation of ROS
(reactive oxygen species) and RNS (reactive nitrogen species).
It further sustains mitochondrial ATP production, even
though at a decreased rate, the electrochemical gradient
(ψ) and other mitochondrial functions like calcium sequestration [16–18]. The decreased ATP production by oxidative
phosphorylation increases cellular dependence on efficient
glucose uptake and increased glycolytic activity [19,20]. Thus
extended or chronic exposure to hypoxia results in genetic
reprogramming of glycolytic enzymes [21].
The NO production during mild-to-moderate hypoxic
conditions may be prolonged or even increased through an
enhanced activity and expression of endothelial NOS (nitric
oxide synthase) [22,23]. Additionally, chronic hypoxia may
lead to an up-regulation of mitochondrial NOS activity [24].
A possible interaction of mitochondrial NOS with complex
IV further increases the regulatory capacity of NO [25].
Besides NOS, nitrite reduction involving xanthine oxidoreductase in the heart [26] and deoxyhaemoglobin in erythrocytes [27] may contribute to the cellular NO levels.
NO and protein nitration in mitochondria
Under normoxic conditions 0.1–3% of the oxygen consumed
by mitochondria is converted to superoxide [28]. During
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Figure 1 Mitochondrial nitration
Crucial enzymes of several mitochondrial metabolic and antioxidant pathways are nitrated including fatty acid β-oxidation
(orange), tricarboxylic acid cycle (pink), amino acid metabolism, ketone body metabolism and respiratory chain (green).
Nitrated proteins are marked in red. A-DH, acyl-CoA dehydrogenase; EH, enoyl-CoA hydratase; HA-DH, hydroxyacyl-CoA
dehydrogenase; KT, β-ketothiolase; F, fumarase; M-DH, malate dehydrogenase; CS, citrate synthase; A, aconitase; I-DH,
isocitrate dehydrogenase; K-DH, α-ketoglutarate (2-oxoglutarate) dehydrogenase; SS, succinyl-CoA synthetase; G-DH,
glutamate dehydrogenase; SCOT, succinyl-CoA:3-oxoacid CoA-transferase; I, II, III, IV, respiratory complexes; ETF, electron
transfer flavoprotein; ETF-Q R, ETF-ubiquinone oxidoreductase; GPx, glutathione peroxidase; mtNOS, mitochondrial NOS; ANT,
adenine nucleotide translocator; VDAC, voltage-dependent anion channel.
hypoxia and reoxygenation, the generation of superoxide
increases with peaks during the transitions between hypoxic
and normoxic conditions [29–31]. Once formed, superoxide
may undergo a near-diffusion-controlled reaction with NO
yielding the highly reactive oxidizing peroxynitrite, which
can, especially in the presence of CO2 /bicarbonate anions, react with protein tyrosine residues forming 3-nitro-L-tyrosine
[32]. The resulting high probability of protein nitration
especially in the mitochondrial matrix [33] is reflected by
the fact that a substantial number of mitochondrial proteins
are nitrated in vivo covering all essential metabolic and antioxidant pathways [34,35] (Figure 1).
Up until recently, tyrosine-nitrated proteins have been
viewed as dead end products that indiscriminately induce
downstream events and are destined for protein degradation.
As a result, protein nitration became a now-established marker for the extent of RNS production and therefore cellular
stress states during both physiological and pathological conditions [36,37]. More recent findings revealed that cumulative
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protein tyrosine nitration may be actively involved in the
onset and/or progression of various diseases [38–41], as well
as in the pathogenesis of acute pathological conditions like
ischaemia–reperfusion [42], both associated with increased
levels of RNS. For example, frequent nitration of MnSOD
(mitochondrial manganese superoxide dismutase) [34,40,43]
may lower the mitochondrial antioxidant potential and contribute to apoptosis and shedding of airway epithelial cells,
leading to airway hyperresponsiveness and remodelling in
asthma [40]. However, the exact effects of protein tyrosine
nitration have not been fully delineated in vivo, since
the levels of nitration of individual proteins and sites of
modification are unknown.
Nitrative signalling
The various potential cellular effects of protein tyrosine nitration, including enzyme activation or inactivation [36,44,45],
raise the issue of whether it might also yield signalling functions. It comprises two principal possibilities, alteration of
Cellular Information Processing
existing signalling pathways like tyrosine phosphorylation–
dephosphorylation and/or a separate pathway based on
tyrosine nitration/denitration. The numbers of studies for
both possibilities are limited. One such study investigating
stimulation of N-methyl-D-aspartate receptor in astrocytes
showed tyrosine nitration of the peripheral-type benzodiazepine receptor and ERK1 (extracellular-signal-regulated
kinase 1) [46]. Furthermore, tyrosine nitration of the p85
subunit of PI3K (phosphoinositide 3-kinase) was induced by
high glucose in retinal endothelia cells, blocking PI3K and
Akt-1 kinase activity [47].
Proof of a nitration–denitration pathway requires the
fulfilment of four basic criteria, i.e. (i) specific modification
of targets, (ii) altered activity/functionality of the modified
protein, (iii) reversibility of the modification, and (iv) nitration/denitration occurring on a physiological timescale [45].
Specificity of protein nitration has been demonstrated in
normal tissues and in a number of tissues from disease
models [34,39,45]. Similarly, nitration of specific proteins has
been shown to alter the functionality. For example, in vitro
exposure to peroxynitrite specifically nitrated the highly
conserved C-terminal Tyr363 residue of aldolase A, with
an additional secondary nitration at Tyr342 and Tyr222 [44].
This nitration led to alterations in the kinetic parameters K m
and V max , and hence reduced the activity. However, in vivo
physiological consequences are not always that clear, as
patients with a partially defective mutant aldolase A show
only a minor impairment of their glycolytic activity. Other
affected proteins in the same pathway can complicate the
biological outcome. Nitration of aldolase A in vivo is often
accompanied by nitration of GAPDH (glyceraldehyde-3phosphate dehydrogenase). When aldolase is nitrated alongside its downstream enzyme GAPDH, a more significant
effect on glycolysis may occur even at low degrees of nitration
as the reaction of aldolase has a small Gibbs energy (G =
−0.72 to −1.3 kJ/mol) for the forward reaction. The small
Gibbs energy suggests that the downstream enzyme must be
capable of high activity to continue the reaction. GAPDH
has been shown to be particularly sensitive to chemical
modifications by peroxynitrite. Alterations of activity for a
number of other proteins including MnSOD [41], succinylCoA:3-oxoacid CoA-transferase [35] and actin [45] have also
been demonstrated.
Reversibility, covering the elimination of the nitro group
from the tyrosine by a denitrase as well as chemical modification of it but not degradation of the nitrated protein, is still
the least well-elucidated criterion [45,48]. Kamisaki et al. [48]
have partially characterized a potential nitrotyrosine denitrase up-regulated in spleen and lung extracts of lipopolysaccharide-treated rats. The activity from extracts greater in
size than 10 kDa, was labile to heat and trypsin treatment,
suggesting that it was protein based. Recently, we demonstrated for the first time that selective protein tyrosine
denitration and renitration, dependent on de novo NO
synthesis, occurs in a tightly regulated manner in mitochondria during recurrent hypoxia–anoxia and reoxygenation
episodes [43,49] (Figure 2). Thereby the extent to which
Figure 2 Nitrotyrosine quantification in mitochondria under
different oxygenated states
Determination of the nitrotyrosine (Y-NO2 ) to tyrosine (Y) ratio by
stable isotope dilution HPLC-tandem MS as previously described in
mitochondria before hypoxia–anoxia (C), after 5–20 min hypoxia–anoxia
in the presence of l-arginine (HA), after 20 min in oxygenated mitochondria in the presence of l-arginine (OX) and after 20 min of reoxygenation following 20 min hypoxia–anoxia (HA-RO) [43]. Significant
(P < 0.05) alterations are marked by an asterisk (*).
both processes take place depends on the duration of the
hypoxic–anoxic phase. While we could reasonably exclude
proteolysis, we could not rule out the reduction of 3nitrotyrosine residues to 3-aminotyrosine. Nonetheless, the
demonstration of a process of controlled loss and gain of
protein nitration, apart from simple proteolysis, controlled
by the oxygen tension of the microenvironment and fulfilling
the other criteria for a signalling pathway, is of particular
interest considering the mitochondrial targets of protein
nitration (Figure 1) and the essential role of mitochondria
in energy production and apoptosis. Selective denitration
of crucial nitrated mitochondrial enzymes depending on
the duration and severity of hypoxic episodes in addition
to the effects of NO and phosphorylation probably helps to
sustain mitochondrial ATP production, ψ, and an increased
antioxidant capacity, which is important for the prevention
of metabolic failure and cell death. In this context, it is also
important to note that the dependence of the cellular damage
caused by reoxygenation after oxygen deprivation in tissues
depends on the severity and duration of hypoxia or anoxia
[43,50].
If protein nitration is indeed important in normal physiological regulation then it is probable that the alterations in
the nitration levels may contribute to different biological
outcomes (Figure 3). While cellular adaptive processes including nitration/denitration and protein degradation by the
20 S proteasome [51] may be designed to cope up with regular
(Figure 3A) and slightly elevated (Figure 3B) degrees of
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Figure 3 Oxidative stress – protein tyrosine nitration
Depending on the degree of oxidative stress resulting from regular
metabolism and pathological conditions, we distinguish three different
situations that determine the nitration/denitration balance: low
metabolic stress (A) with dominating balance between nitration
and denitration; increased stress (B) with enhanced protein nitration
rate exceeding denitration; and excessive stress (C) with a protein
nitration rate exceeding denitration and protein degradation leading to
accumulation and aggregation of nitrated proteins.
oxidative stress, excessive oxidative stress (Figure 3C) could
lead to overwhelmed denitration, accumulation of different
nitrated proteins, including those not nitrated before, and
protein aggregation. As with hyperphosphorylation, which
can lead to detrimental protein function shown for tau
protein in Alzheimer’s disease [52], excessive or inappropriate nitration can also lead to disease or acute pathological
conditions through the dysregulation of metabolic, regulatory and antioxidative pathways.
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This work was supported by grants from the American Heart
Association (0325313B), National Institutes of Health grants
HL076491 and CA53919 as well as the Alpha-1 Foundation (P102-6).
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