Post-translational modifications of superoxide dismutase

Biochimica et Biophysica Acta 1804 (2010) 318–325

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a p a p

Review

Post-translational modifications of superoxide dismutase Fumiyuki Yamakura a,b,⁎, Hiroaki Kawasaki b a b

Department of Chemistry, Juntendo University School of Health Care and Nursing, Japan Institute of Environmental Medicine and Gender Specific Medicine, Juntendo Graduate School of Medicine, Inba, Chiba 270-1606, Japan

a r t i c l e

i n f o

Article history: Received 16 August 2009 Received in revised form 5 October 2009 Accepted 6 October 2009 Available online 22 October 2009 Keywords: Post-tanslational modification Superoxide dismutase Nitration Phosphorylation Glutathionylation Metal misincorporation

a b s t r a c t Post-translational modifications of proteins control many biological processes through the activation, inactivation, or gain-of-function of the proteins. Recent developments in mass spectrometry have enabled detailed structural analyses of covalent modifications of proteins and also have shed light on the posttranslational modification of superoxide dismutase. In this review, we introduce some covalent modifications of superoxide dismutase, nitration, phosphorylation, glutathionylaion, and glycation. Nitration has been the most extensively analyzed modification both in vitro and in vivo. Reaction of human Cu,Zn superoxide dismutase (SOD) with reactive nitrogen species resulted in nitration of a single tryptophan residue to 6-nitrotryptophan, which could be a new biomarker of a formation of reactive nitrogen species. On the other hand, tyrosine 34 of human MnSOD was exclusively nitrated to 3-nitrotyrosine and almost completely inactivated by the reaction with peroxynitrite. The nitrated MnSOD has been found in many diseases caused by ischemia/reperfusion, inflammation, and others and may have a pivotal role in the pathology of the diseases. Most of the post-translational modifications have given rise to a reduced activity of SOD. Since phosphorylation and nitration of SOD have been shown to have a possible reversible process, these modifications may be related to a redox signaling process in cells. Finally we briefly introduce a metal insertion system of SOD, focusing particularly on the iron misincorporation of nSOD, as a part of posttranslational modifications. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Post-translational modifications play a major role in regulating proteins, possessing a wide variety of mechanisms that include changes in protein activities, interactions, and subcellular localizations. More than 200 varieties of post-translational modifications have been identified. Recent developments of proteomic analyses for detection of the modified sites in proteins, such as a combination of 1st or 2nd dimensional PAGE, specific polyclonal and monoclonal antibodies for the modification site, and/or LC-MS/MS analysis, have enabled the identification of new types of modifications and the new proteins having the specific modifications in vitro and in vivo. Furthermore, application of these proteomic analyses for whole protein mixtures isolated from cells, tissues, or organs from patients or disease-model animals enables us to analyze alterations of the post-translational modifications under pathophysiological conditions. Oxidative modification and glycation of superoxide dismutase (SOD)

were early examples of post-translational modifications of SOD [1,2]. Applications of the newly developed proteomic analyses have brought us findings regarding the new post-translational modifications of SODs. Recently, superoxide and its related reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been found to affect not merely toxic species for cells but also to serve as possible modifiers of cell signal transductions [3,4]. Therefore, regulation of SOD activity by the post-translational modifications could represent a new field of interest regarding the cell signal process. In this review, we summarize several post-translational modifications of SODs; nitration, phosphorylation, glutathionylation, glycation, and metal misincorporation to apoMnSOD; and discuss their pathophysiological and physiological significance. 2. Nitration 2.1. Generation of reactive nitrogen species

Abbreviations: ADR, adriamycin; I/R, Ischemia/reperfusion; iNOS, inducible nitric oxide synthase; eNOS, endotherial nitric oxide synthase; nNOS, neuronal nitric oxide synthase; PAGE, polyacrylamide gel electrophoresis; RNS, reactive nitrogen species; SOD, superoxide dismutase ⁎ Corresponding author. Department of Chemistry, Juntendo University School of Health Care and Nursing, Japan. E-mail address: [email protected] (F. Yamakura). 1570-9639/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2009.10.010

Reactive nitrogen species (RNS), such as, peroxynitrite (ONOO−) and nitrogen dioxide (NO2), have been implicated as causes of various pathophysiological conditions, including inflammation, neurodegenerative and cardiovascular diseases and cancer [5–7]. These species act as nitrating agents that affect protein function [5–7]. Peroxynitrite is produced by the radical-radical coupling

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reaction between superoxide anion (OU2) and nitric oxide (NO) with a rate-limiting rate constant and it is known to cause oxidation and nitration of proteins, unsaturated fatty acids, nucleotides, small biomolecules, such as tyrosine and tryptophan [5–8]. In the presence of physiological concentration of carbon dioxide, peroxynitrite is known to form a peroxynitrite–carbon dioxide adduct, which exhibits more reactivity to nitrate biomolecules than peroxynitrite (Fig. 1) [5–7]. Another RNS, NO2, is produced by the oxidation reaction of nitrite, which is catalyzed by peroxidases in the presence of hydrogen peroxide (Fig. 1) [5–7]. A specific nitration product of tyrosine, 3-nitrotyrosine, has been widely used as a biomarker for the RNS production in vivo. The consequences of nitration of tyrosine residues in proteins include altered function and structure of the protein, modulation of the catalytic activity of enzymes (inactivation or activation), susceptibility to proteolysis and/or affecting signal transduction by hindering tyrosine phosphorylation [4–7,9]. 2.2. Nitration of Cu, ZnSOD Nitration is one of the most extensively studied modifications of SODs. Formation of 3-nitrotyrosine residues by the reaction with peroxynitrite was first reported for bovine Cu, ZnSOD by Ischiropoulos et al. [10]. Crystal structure of peroxynitrite-modified bovine Cu, ZnSOD clearly revealed Tyr-108 was modified to 3nitrotyrosine [11]. The modified enzyme showed the same activity as that of the native enzyme [10]. In contrast to bovine Cu, ZnSOD, human Cu, ZnSOD has no tyrosine residue but has a single tryptophan residue (Trp32) in its amino acid sequence. By means of MS and HPLC-photodiode array analyses, we found that reaction of peroxynitrite–CO2 and myeloperoxydase/hydrogen peroxide/nitrite system with human recombinant Cu, ZnSOD resulted in the formation of 6-nitrotryptophan as a major nitration product [12,13]. Several other oxidized products of tryptophan (kynurenine, oxindole-3-alanine, dihydroxytryptophan) were also observed. After this finding, 6-nitrotryptophan was found to be a major nitrated product of tryptophan residues in other proteins reacted with peroxynitrite/CO2 or myeloperoxydase/H2O2/NO− 2 system [14–17]. We proposed that 6-nitrotryptophan could be a new biomarker for formation of RNS in vivo [12,13]. The modified human Cu, ZnSOD showed partial losses of the enzymatic activity of 30% for peroxynitrite modification and 15% for the myeloperoxydase system modification, respectively [12,13]. In contrast, Alvarez et al. [18] reported that human Cu, ZnSOD lost most of the enzymatic activity (90%) by peroxynitrite treatment and found to form histidinyl radical. They did not use carbon dioxide in their reaction system,

Fig. 1. Major pathways to generate nitrotyrosine- and nitrotryptophan-containing proteins. Red arrows: up regulation.

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therefore hydroxyl radical-like species derived from peroxynitrite homolysis likely to be involved. Regarding Trp32 in human Cu, ZnSOD, Taylor et al. [19] reported that W32F mutant of Cu, ZnSOD decreased both the cytotoxicity of G93A mutant SOD and its propensity to form cytoplasmic inclusions in neurons in mouse model of familial amyotrophic lateral sclerosis (FALS). This evidence suggests a possibility that the nitration of Trp32 could also have some effect on the cytotoxicity of this protein in FALS. 2.3. Nitration of Fe/MnSOD Tyrosine nitration in human MnSOD was reported by MacMillanCrow et al. [20] for the first time in chronic rejection of human renal allograft. Total MnSOD protein was increased but the enzyme activity was decreased. Reaction of peroxynitrite with a recombinant human MnSOD resulted in a decrease of enzymatic activity concomitant increase in tyrosine nitration [21]. They found that three tyrosine residues (Y34, Y45, Y193) of the nine total tyrosine residues were nitrated by MS analysis [21] and suggested that not only the active site Tyr34 but also two other residues could contribute to complete inactivation by using Y34F MnSOD mutant, in which the mutant enzyme has been completely inactivated by peroxynitrite [22]. They pointed out that formation of dityrosine as an oxidized product of tyrosine residues could also contribute to the inactivation of the enzyme, and especially more efficiently so in the mutant enzyme [21,22]. In contrast, we found that Tyr34 was the only residue nitrated with peroxynitrite by using MS analysis and that the activity was lost concomitant with a rate of the tyrosine residue nitration [23,24]. This residue is located within ∼ 5.5 Å from manganese in the active site. Quijano et al. [25] also found the addition of a single nitro group to MnSOD and loss of most of the enzymatic activity. The crystal structure of nitrated human MnSOD has been determined by Quint et al. [26]. They showed that about 75% of Tyr 34 was nitrated without nitration of other tyrosine residues and that the enzymatic activity was inhibited nearly 80%. In addition, Neumann et al. [27] prepared 3-nitrotyrosine incorporated human MnSOD at the position 34 by using an aminoacyl-tRNA synthetase/tRNA pair for the cotranslational, site-specific incorporation of 3-nitrotyrosine at genetically encoded sites. They found that the quantitative nitration of tyrosine 34 of human MnSOD inactivated up to 97% of its enzymatic activity. Together, we can conclude that peroxynitrite nitrates tyrosine 34 almost exclusively and that nitration of tyrosine 34 alone is sufficient to relieve the enzymatic activity of human MnSOD. Peroxynitrite is known to react with protein metal centers first and accelerate nitration of adjacent tyrosine residues in a metal-center-dictated nitration mechanism [5,6]. This mechanism was proposed for the nitration of tyrosine 34 of MnSOD [25] and could explain the specific reactivity of tyrosine 34 compared with other eight tyrosine-residues in the subunit. Three possible mechanisms for inactivation by the modification have been proposed; namely, steric interference for substrate access and binding, a possible weakening of hydrogen-bond network that supports proton transfer in catalysis, and electrostatic effects related to the presence of the nitro group and the resulting change of the redox potential [24–28]. Although one contradictory result has been reported for Escherichia coli FeSOD [29], Ischropoulos et al. [10] and we [24] have observed inactivation of Fe-SOD from E. coli and Pseudomonas ovalis, respectively, by peroxynitrite dose-dependently. Recently, Larrainzar et al. [30] observed that recombinant FeSOD from a plant cell, Vigna unguiculate, was nitrated and inactivated by incubation with SIN-1, which simultaneously generated NO and O− 2 . Therefore, we conclude that RNS could nitrate and inactivate most of the Fe and MnSOD; probably through the nitration of the critical Tyr34 (human MnSOD numbering) residues of the enzymes.

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2.4. Nitration of mitochondrial MnSOD in diseases 2.4.1. Kidney Nitrated MnSOD was first found in rejected human renal allograph as shown above [20]. Tyrosine nitration of MnSOD and cytochrome c was observed at the earliest time point as an event which preceded significant renal injury in a renal ischemia/reperfusion (I/R). This provides a model to analyze the time course of events during renal transplantation [31,32]. ATP depletion, MnSOD inactivation, nitrotyrosine formation, and renal dysfunction in the renal I/R model rat were prevented by pretreatment of a catalytic antioxidant, Mn(III) meso-tetrakis (N-n-hexylpyridinium-2-yl) porphyrin [33]. This evidence suggests a central role of MnSOD in renal I/R injury. Cold preservation of cadaveric kidneys for renal transplantation is known to cause damage during both the preservation episode and the reperfusion phase. Significant renal injury, nitrotyrosine production, inactivation of MnSOD, and subsequent reduced activities of the mitochondrial respiratory complexes have been observed in a rat model of in vivo renal cold preservation followed by warm reperfusion [34]. Nitration of MnSOD is also expected in this case. Angiotensin II-induced hypertension is associated with increase in superoxide production by the activation of NADPH oxidase [35]. MnSOD from angiotensin IIinfused rat kidney demonstrated both significant increase in tyrosine nitration and decrease in its enzymatic activity [36]. Quantitative assessment of nitrated MnSOD was attempted in this study and showed a 13-fold increase of 3-nitrotyrosine content of kidney MnSOD after angiotensin II infusion [36]. Nitrated MnSOD is usually detected by a combination of anti 3-nitrotrysine antibody and anti MnSOD antibody. Xu et al. [37] developed a sequence-specific antibody toward chemically synthesized peptide within the active site of human MnSOD sequence containing Tyr34 (25LHHSKHHAA[nY]VNNLNV40) and used histochemistry and immunoblotting to demonstrate MnSOD nitration in rat kidney with angiotensin II infusion. Tyr 34 nitrated MnSOD was also observed by immunostaining of kidney tissue from streptozotocin-induced diabetic apolipoprotein E-deficient mice by using the same sequence-specific antibody [38]. These results confirm that the specific site of tyrosine nitration first detected in vitro studies (Tyr34) is also observed in these pathological situations. 2.4.2. Heart and vascular system Nitration and inactivation of MnSOD was also found in acutely rejecting cardiac allografts [39]. A potential major source of this nitration could be iNOS-dependent pathway. Cyclosporine A has remained as a crucial immunosuppressant with a major therapeutic role in solid organ transplantation. However serious toxic effects, such as nephrotoxicity and vascular injury, have been associated with its clinical use. Navarro-Antolin et al. [40] found formation of nitrated MnSOD in cultured bovine aortic endothelial cells when exposed to cyclosporine A. This may represent a pathogenetic mechanism of the vascular injury. Vascular aging is mainly characterized by endothelial dysfunction. Free NO level was decreased in aged rat aorta concomitant with higher expression of eNOS and enhanced O− 2 production. Nitrated MnSOD was demonstrated in rat aorta with increased age [41]. 2.4.3. Brain and neuronal cells Alzheimer's disease is a multifactorial, progressive, age-related neurodegenerative disease. Inactivation and nitration of MnSOD was observed with age in brain homogenates of presenilin mutant's double knock-in mice, which showed deposition of Aβ as well as an increase in the levels of insoluble Aβ1–40/1–42. Mitochondrial respiration also decreased in the knock-in mice. Nitration and inactivation of MnSOD was observed, which may have been caused by increased Aβ and an associated decrease in mitochondrial function was also observed [42]. Similar results of MnSOD nitration and inactivation were also observed in primary culture of mature neuron

cells from amyloid precursor protein and presenilin1 knock-in mice [43]. Nitrated MnSOD was observed in cerebrospinal fluid of 66 patients with neurogenic disease. The nitrated MnSOD levels were strikingly elevated in amyotrophic lateral sclerosis patients and were slightly increased in Alzheimer's and Parkinson's disease patients [44]. Brains of mice and humans after traumatic brain injury demonstrated both tyrosine nitration of MnSOD and dramatic decrease in its enzymatic activity. nNOS, but not iNOS or eNOS, was responsible for MnSOD nitration after the injury in this system [45]. In contrast, membrane lipid peroxidation, protein nitration, and neuronal death after focal cerebral ischemia were significantly reduced in human MnSOD overexpressed transgenic mice, suggesting a pivotal role of MnSOD in the prevention of degenerative disorders of the brain [46]. Some cancer patients receiving adriamycin (ADR) treatment develop a transient memory loss and inability to handle complex tasks, which are referred to as chemobrain by patients. Tangpong et al. [47] demonstrated that treatment of ADR in mice led to an increased circulating level of TNF-α and to nitration and inactivation of MnSOD. Mitochondrial respiration was also declined. They proposed that ADR treatment led to TNF-α mediated iNOS induction to produce NO in brain tissues that subsequently led to a decline in mitochondrial respiration and also led to formation of peroxynitrite. This mechanism was further confirmed by using primary cultures of glia cells isolated from wild-type mice and iNOS knock-out mice [48]. 2.4.4. Other diseases Pulmonary MnSOD was both nitrated and inactivated following hepatic I/R and has been associated with acute lung injury [49]. This evidence suggests that the nitration of MnSOD may contribute to I/Rinduced lung injury and provide a therapeutic target in attenuating multi-system injury following hepatic I/R. Mallery et al. [50] demonstrated that a nitrative stress occurred in situ within patients' AIDS-related Kaposi's sarcoma (AIDS-KS) lesions and also showed that cultured AIDS-KS cells derived from these tumors demonstrated inactivation and nitration of MnSOD. Gastro-esophageal reflux disease is one of the most troublesome and frequent gastrointestinal disorders of the Western world, and may lead to esophageal cancer. A decrease in SOD activity leading to increased mucosal levels of superoxide and peroxynitrite may contribute to the development of esophageal damage and Barrett's esophagus in patients with gastroesophageal reflux. Nitrated MnSOD was observed in patients with esophagitis and Barrett's esophagus [51]. Pathophysiological significance of nitration of Tyr 34 of MnSOD in these diseases could be elucidated as follows (Fig. 2); a high concentration of NO (5–200 μM), which is produced mostly by iNOS induction, can penetrate mitochondrial membrane and may enhance O− 2 production by inhibition of cytochrome oxidase with NO [52]. Since the reaction rate constant between O− 2 with NO is several times faster than that with MnSOD, NO will compete with MnSOD for mitochondrially produced O− 2 to form peroxynitrite. Inactivation of mitochondrial MnSOD by peroxynitrite leads to accumulation of O− 2 and as a consequence more accumulation of peroxynitrite. Both accumulated O− 2 and peroxynitrite may cause oxidative and nitrative damages of mitochondrial components and subsequently result in mitochondrial dysfunction leading to apoptosis or necrosis of cells (Fig. 2) [20,21,23,25,26]. Although it is difficult to clarify whether the nitration and inactivation of MnSOD are causes or consequences in each of the diseases, these phenomena should have pivotal roles, at least in the state of the diseases, since the MnSOD model complex and overproduction of MnSOD could prevent some of these diseases [33,46]. 2.5. Is nitration of MnSOD a reversible process? Although tyrosine nitrated MnSOD was found mostly in oxidative damaged tissues, it is worth considering the possibility that it may

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Fig. 2. Nitration and inactivation mechanism of human MnSOD in mitochondria. Formation of O− 2 and NO is up-regulated, which results in peroxynitrite formation, under pathophysiological conditions, such as ischemia/reperfusion and inflammation. Protonated form of peroxynitrite and NO can cross mitochondrial membrane into matrix space. Under inhibition of cytochrome oxidase by NO, O− 2 is generated by electron transport chain [52], which is reacted with NO to form peroxynitrite. Nitration and inactivation of MnSOD by peroxynitrite leads to accumulation of O− 2 and as a consequence more accumulation of peroxynitrite. This negative cycle may cause mitochondrial impairment catastrophically.

regulate the nitration of MnSOD. It has been suggested that protein 3nitrotyrosine can be reversibly transformed to tyrosine by an enzymatic process without the participation of proteolytic pathways in spleen and lung homogenate [53], brain and heart homogenate [54], or LPS-treated RAW 264.7 cells with Histone H1.2 as a suitable substrate [55]. Denitration of nitrated glutamine synthase and human smooth muscle L-type calcium channels were observed by using spleen protein preparation from LPS-treated rats and cell lysate from LPS-activated RAW 264.7 cells, respectively [56,57]. All of these studies were based on the disappearance of the reactivity of nitrotyrosine-specific antibody for the substrate proteins. Recently, Smallwood et al. [58] proved that calmodulin is another specific substrate for denitrase activity from LPS-activated macrophages by proteomic analysis using LC-MS/MS technique. They also proved that substrate specificities of the denitrase activity were very high and confirmed that the product of the denitrase was native tyrosine residue, not aminotyrosine. A presence of the nitration-denitration process has been reported for mitochondrial MnSOD. A disappearance of the nitrotyrosine-immunoreactive proteins of mitochondria from rat liver, in which MnSOD was included along with seven other proteins, during hypoxia or anoxia treatment and a reversible appearance of the immunoreactivity of those proteins in response to reoxygenation was observed [59,60]. Since protease inhibitor was not effective for this process and substantial decrease in the immunoreactivity occurred within a few minutes, decrease in the nitrotyrosine immunoreactivity was proposed likely performed by a chemical or enzymatic denitration process [59,60]. This evidence suggests a presence of nitration reaction of MnSOD in mitochondria under oxygenated state. Furthermore, although the detailed mechanism and the specific proteins catalyze this process need to be clarified, it is very appealing to suggest that mitochondrial denitration-process (probably corresponding to reactivation-process) of MnSOD may occur in a beneficial adjustment of mitochondrial antioxidant defense during short periods of hypoxia or anoxia [59,60]. They suggested that this event could be a model for tissue ischemia/reperfusion.

3. Phosphorylation Phosphorylation of serine/threonine residues or tyrosine residues is one of the typical translational modifications of proteins. Transient phosphorylation of cytoplasmic Cu, ZnSOD was the first reported by Csar et al. [61] after treatment of myeloid cells by the granulocyte colony stimulating factor (G-CSF). They suggested diminishing of Cu, ZnSOD levels and activity by the treatment with G-CSF, possibly due to an increase in proteolytic clearance of Cu, ZnSOD caused by the phosphorylation. In this case, decrease in SOD level and resulting increase in O− 2 level may contribute modulation of signal transduction event caused by G-CSF in myeloid cells [61]. Archambaud et al. [62] reported on the phosphorylation of bacterial SOD. MnSOD from the cytoplasmic Listeria monocytogenes was phosphorylated on serine and threonine and was found to be less active when bacteria reach the stational growth phase. The most active and nonphosphorylated form of MnSOD was secreted in bacterial culture. This could be a virulence factor of this bacterium, in which secreted MnSOD could counteract the role of host phagocytes. On the other hand, they observed that the secreted MnSOD was phosphorylated and down-regulated in infected cells, which were probably mediated by an unknown cellular kinase. This mechanism could be a new strategy used by host phagocytes to control intracellular infection [62]. Phosphorylated FeSOD in Gramnegative bacterium, Campylobacter jejuni, was also observed by phosphoproteome analysis [63]. Mitochondrial MnSOD is also predicted as a target of phosphorylation. Phosphorylated MnSOD was first reported in potato mitochondria by 2nd-dimensional PAGE /MS analysis using [γ-32P]-ATP but no activity study was done [64]. Hopper et al. [65] reported phosphorylated MnSOD in isolated pig hearts mitochondria by phosphoproteome technique. Furthermore, Ca2+-induced dephosphorylation of MnSOD in isolated mitochondria, which associated with an approximately 2-fold maximum increase in the enzymatic activity, was observed. This evidence suggests that MnSOD activity may be controlled to regulate a steady-state level of superoxide or hydrogen peroxide in matrix by Ca2+ dependent signaling processes. Recently a possible

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phosphorylation site of rat mitochondrial MnSOD was determined as Ser82 by the incubation of S82A mutant MnSOD with mitochondrial protein extracts from rat neuroblastoma cells and [γ-32P] ATP [66], though an activity study was not done. Taken together, phosphorylation could represent a novel regulation mechanism for SODs from bacteria to vertebrates. Although disease related phosphorylation of SOD has not reported yet, further studies of this field may lead to a fine-tuning of the SOD function for oxidative damage and redox signaling of the cells. In order to clarify entire figure of the regulation mechanism, identification of SODspecific kinases and phosphatases, phosphorylation site of SODs, and more detailed analysis on the changes of SOD functions by phosphorylation remain to be investigated. 4. S-glutathionylation S-glutathionylation is the specific post-translational modification of protein cysteine residues by the addition of glutathione and is promoted by oxidative or nitrosative stress and also occurs in unstressed cells. It can regulate a variety of cellular processes by modulating protein function and to prevent irreversible oxidation of protein thiols [67]. Glutathionylated human Cu, ZnSOD at Cys111 was isolated from human erythrocytes. The glutathionylation of Cys111 showed no significant activity change but promoted Cu, ZnSOD mutants-mediated, which has much less enzymatic activity, with a 2-fold increase in the Kd [68]. The dimer dissociation of human Cu, ZnSOD is known to be the first step to make aggregation form, which is known to be involved in Cu, ZnSOD mutants-mediated familial amyotrophic lateral sclerosis (FALS); a fatal disease characterized by progressive motor neuron loss [69]. Therefore they proposed that the glutathionylation of human Cu, ZnSOD resulted in a reduction of its enzymatic activity and promotion of its aggregation. The later event could possibly link to FALS by a facilitating of Cu, ZnSOD aggregation, which is an early event for SOD mutants-linked FALS [69]. FeSOD from Phychrophilic eubacterium Pseudoalteromonas haloplankis (Ph) was isolated as an S-glutathionylated form at the site of Cys57, which is a single and highly reactive cysteine residue. The glutathionylation of PhSOD occurred in a growing culture of Ph and during its overproduction in E. coli cells. The specific activity did not varied more than 10% compared with that of the untreated enzyme. However, this modification in PhSOD protected the enzyme from tyrosine nitration and peroxynitrite inactivation and was enhanced upon cell exposure to oxidative agents [70]. Since Ph is well adapted to protection against reactive oxygen species under cold conditions, a function of this modification was proposed as a cold-adaptation strategy to improve the antioxidant cellular defense mechanism [70]. They also found s-glutathionylation of recombinant rat MnSOD grown in E. coli, but no activity study was done in this case [65]. 5. Glycation Glycation is thought to be involved in structural and functional changes of proteins and to occur during normal ageing with accelerate rates in patients with diabetes mellitus. Glycation of Cu, ZnSOD is one of the earliest findings for a post-translational modification of SODs [2,71]. Glycation in Cu, ZnSOD may cause fragmentation of the enzyme [72], and loss of the enzymatic activity may lead to physiological problems in diabetes patients [73]. Although we have many studies on glycation of Cu, ZnSOD, we will not show further details in this review since we already have excellent reviews on the glycation of Cu, ZnSOD [73,74]. The effects of four post-translational modifications; nitration, phosphorylation, s-glutathionylation, and glycation on SOD activities are summarized in Fig. 3.

Fig. 3. Activity changes of SODs by the post-translational modifications. Downward arrows indicate decrease of the enzymatic activity of each SODs. Among them, dashed arrows represent the effect of the modifications on the activities was limited to a partial loss. Arrows covered by squares show the modifications, which have been found in vivo. Horizontal arrow indicates no change on the enzymatic activity by the modification, but prevention of peroxynitrite-dependent inactivation by this modification was reported [68].

6. Metal misincorporation of MnSOD—when angel metamorphoses into damon Insertion of the catalytic metals to SOD is one of the posttranslational modifications of SOD. Detailed studies on the mechanism of copper insertion into cytosolic Cu, ZnSOD, which is facilitated by the specific copper chaperone for Cu, ZnSOD (CCS), have been achieved by Culotta's group. Many excellent reviews are available for this field [75–78]. The incorporation system of the catalytic metal to mitochondrial MnSOD is totally different from that of Cu, Zn-SOD. MnSOD generally resides in the matrix of the mitochondria in eukaryotes. Polypeptide of mitochondrial MnSOD is encoded by a nuclear gene with precursor polypeptide. After it is imported into the mitochondrial matrix, the pre-sequence of the enzyme is cleaved and manganese is subsequently inserted into apo-MnSOD. Metal activation process requires translation of newly synthesized apo-MnSOD into mitochondria. Two membrane transporters have been identified as facilitators of manganese insertion to apo-MnSOD by yeast studies. The first one is Smf2p manganese transporter, which is one of two Nramp metal transporters that function in manganese uptake and trafficking in yeast [79]. Smf2p is important for cell surface uptake of manganese. In smf2 mutant, MnSOD polypeptides were found to be accumulated in the mitochondria in an iron-substituted form [80]. The other one is Mtm1p, a member of the mitochondrial carrier family of transporters, which all reside in the mitochondrial inner membrane and function to exchange solutes between the mitochondria and cytosol [81]. MnSOD activity was also lost in mtm1Δ cells but could not be restored by the supplementing of manganese to the growth medium. On the contrary, mtm1 Δ mutants accumulated somewhat higher than normal levels of mitochondrial manganese and also very high levels of mitochondrial iron [81]. Therefore Mtmp1 is not the manganese transporter for the mitochondria but may transport some unidentified factor, which may control the bioavailability of manganese and iron for mitochondrial MnSOD [81]. Iron incorporated MnSOD was also formed in mtm1 Δ mutants, in which accumulated iron may be changed to apoMnSOD available form, out-compete to manganese [80]. We focus the rest of this review on a possible biological action of the iron misincorporated MnSOD. X-ray structural studies have shown that FeSODs and MnSODs have a large degree of sequence homology, similar threedimensional structure and the same amino acid residues as ligands with similar geometry for its metals [82]. However, most of FeSOD and MnSOD from bacteria require original metals for the activity [83,84], although these can bind wrong metals with high affinity. A few exceptions have been found in some bacteria which use both metals to exhibit the enzymatic activity called cambialistic SOD

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[85]. Recently, we demonstrated that human mitochondrial MnSOD also exhibited as high a metal-specificity as those of other bacterial MnSOD by in vitro metal-substitution study [86]. The inactive Fesubstituted MnSOD (Fe-MnSOD) showed more thermal stability than that of native MnSOD and a hydrogen peroxide-mediated radical-generating activity [86] the same as that which has been found in Cu, ZnSOD [87,88]. This reactivity could be a new gain-offunction of the Fe-MnSOD. Therefore Fe-MnSOD in mitochondria, as present in mtm1 mutant or smf2 mutant of baker's yeast [79–81], may produce a disadvantage for oxidative stress in three ways; not only a loss of the enzymatic activity but also a gain of radicalgenerating ability and increased stability of Fe-MnSOD. Although presence of Fe-MnSOD has not been confirmed yet, iron overload in mitochondria has been shown in several diseases. X-linked sideroblastic anemia with ataxia is caused by mutation of human abc7 gene and observed in mitochondrial iron overloading [89,90]. Silencing of ABCB7 gene in HeLa cells by siRNA resulted in an accumulation of iron in mitochondria and MnSOD inactivation [91]. Hereditary ferritinopathy (neuroferritinopathy), which is related to insertional mutation in the ferritin light chain gene (FTL), has been shown to cause abnormality to mitochondria in dentate neurons. It has been proposed that this may be due to an imbalance in iron homeostasis in mitochondria [92]. Since misincorporation of iron to MnSOD is likely raised in these diseases as it was found in the yeast mutant [80], the finding of Fe-MnSOD in these diseases is very important and interesting subject to be investigated. If misincorporation of iron to MnSOD should in fact proceed in these diseases, it could be a cause of these diseases through mitochondrial damage caused by the three toxic properties of Fe-MnSOD. 7. Conclusions and perspectives Superoxide dismutase catalyzes the dismutation reaction of superoxide anion into oxygen and hydrogen peroxide, which is in turn decomposed into water by glutathione peroxidase, peroxiredoxin, and catalase. Superoxide anion itself has limited reactivity for biomolecules, which are oxidation and decomposition of iron– sulfur cluster of several enzymes [93] and reaction with NO to produce very reactive and toxic species, peroxynitrite (Fig. 4) [5–7]. Inactivation or down-regulation of SOD activity causes an increase

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of steady state concentration of superoxide, which could enhance formation of peroxynitrite. Resulting high steady state concentration of peroxynitrite cause cell injury and could be a pivotal factor to cause various diseases. Hydroxyl radical is another toxic reactive species, which is produced by the reduced-metal catalyzed oneelectron reduction of hydrogen peroxide, which is a reaction product of SOD (Fig. 4). An increase of steady-state concentration of hydrogen peroxide may be caused by cases such as an independent up-regulation of superoxide dismutase activity, subsequently that could induce an increase formation of hydroxyl radical in cooperation with reduced metals (Fig. 4). Recently, a modulation of the activities of some proteins in signal transductions, such as NF-κB and protein tyrosine phosphatases, by hydrogen peroxide through a direct or indirect oxidation of specific cysteine residues in the proteins has been reported [3,94]. Superoxide and hydrogen peroxide also react with iron– sulfur cluster of cytosolic iron regulatory protein-1 (IRP-1) to form apo-protein, which could bind the specific mRNA. This mRNA is known as an iron-responsive element and produce iron-uptake protein in low oxidation stress conditions [3,93]. Furthermore, recent studies suggest the possibility that peroxynitrite also may modulate signal transduction pathways through a blocking of tyrosine phosphorylation sites by nitration and regulating activities of various kinases and phosphatases [4] (Fig. 4). The evidence reported above supports the concept that an increase in steady state concentration of superoxide, hydrogen peroxide, and peroxynitrite may contribute modulation of cell signals in some controlled situations. Since superoxide is located the upstream of ROS and peroxynitrite forming pathways, SOD activity could affect the concentration of superoxide, hydrogen peroxide, and peroxynitrite. Although many other factors are also likely to be involved in controlling the steady state concentration of these species, such as O− 2 generating enzymes, NO synthases, and glutathione peroxidase, peroxiredoxin, and catalase, the modulation of SOD activity by the post-translational modifications could prove to be a significant part in the controlling of these species. Therefore, further studies on the reversible process of the post-translational modification of SODs and on the specific enzymes, which control phosphorylation, nitration, and glutathionylation, will be future important targets that may elucidate a possible participation of SOD for the signal transduction pathways.

Fig. 4. Toxic and signal transduction functions of superoxide-related species. Red arrows represent the effects of down-regulation of SOD activity on the steady state concentration of superoxide and its related species. Blue arrows represent the opposite case. In this figure, it is assumed that activities of GPx (glutathione peroxidase), Prx (peroxiredoxin), and catalase are not changed. PTP; protein tyrosine phosphatase, IRP; iron-responsive protein.

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Acknowledgements We thank Professor Bruce E. Allen for his critical manuscript review. This study was supported by Grants-in-Aid for Scientific Research from the Society for Promotion of Science, Japan (18500514) and by the “High-Tech Research Center” Project for Private Universities: matching fund subsidy from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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