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.
- nitric oxide
- oxidative stress
- tyrosine 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 . Protein phosphorylation provides crucial reversible adjustments for sensing and regulating ATP flux. It contributes to the activation of the AMP-dependent protein kinase pathway  and regulates the activities of key enzymes like pyruvate kinase [7,8], pyruvate dehydrogenase  and 6-phosphofructo-2-kinase/fructose bisphosphatase 2 , 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 FoF1ATP synthase . Thus phosphorylation takes part in adjustment of rate, yield and source of cellular ATP production by modulating the activities of the oxygen-dependent mitochondrial oxidative phosphorylation as well as glycolysis .
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 . 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 . 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 (Δψ) 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 .
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 . A possible interaction of mitochondrial NOS with complex IV further increases the regulatory capacity of NO . Besides NOS, nitrite reduction involving xanthine oxidoreductase in the heart  and deoxyhaemoglobin in erythrocytes  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 . During 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 . The resulting high probability of protein nitration especially in the mitochondrial matrix  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 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 , 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 . 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.
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 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) . 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 .
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 . 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 . This nitration led to alterations in the kinetic parameters Km and Vmax, 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-3-phosphate 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 (ΔG=−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 , succinyl-CoA:3-oxoacid CoA-transferase  and actin  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.  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 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 3-nitrotyrosine 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, Δψ, 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  may be designed to cope up with regular (Figure 3A) and slightly elevated (Figure 3B) degrees of 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 , excessive or inappropriate nitration can also lead to disease or acute pathological conditions through the dysregulation of metabolic, regulatory and antioxidative pathways.
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).
Cellular Information Processing: A Focus Topic at BioScience2005, held at SECC Glasgow, U.K., 17–21 July 2005. Edited by F. Antoni (Edinburgh, U.K.), C. Cooper (Essex, U.K.), M. Cousin (Edinburgh, U.K.), A. Morgan (Liverpool, U.K.), M. Murphy (Cambridge, U.K.), S. Pyne (Strathclyde, U.K.) and M. Wakelam (Birmingham, U.K.).
Abbreviations: Δψ, 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
- © 2005 The Biochemical Society