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Superoxide as inhibitor of calcineurin and mediator of redox regulation
Toxicology Letters 139 (2003) 107 /110 www.elsevier.com/locate/toxlet
Short communication
Superoxide as inhibitor of calcineurin and mediator of redox regulation Volker Ullrich *, Dmitry Namgaladze, Daniel Frein Mathematisch-Naturwissenschaftliche Sektion, Fachbereich Biologie, Universita¨t Konstanz, D-78457 Konstanz, Germany
Abstract The concept of NO as a redoxactive messenger has to be broadened by including superoxide as an antagonistic messenger. Superoxide alone was found to inhibit calcineurin by interacting with the FeII /ZnII binuclear site. This links oxidative stress conditions with a Ca-dependent phosphorylation/dephosphorylation cascade. When NO and superoxide are generated at equal fluxes the resulting peroxynitrite can cause tyrosine nitrations (e.g. prostacyclin synthase inhibition) or oxidations of zinc-fingers in proteins, indicating a new messenger function. Finally, if generated in excess, NO can convert peroxynitrite to N2O3 as a nitrosating agent. Thus, the NO/superoxide system provides four different messengers affecting important regulatory pathways. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Nitrogen monoxide/superoxide system; Peroxynitrite; Calcineurin
1. Introduction Nitrogen monoxide (nitric oxide, NO) has important functions as a signaling molecule but no clear-cut concept is available for its physiological effects since it has been described as a ‘double-edged sword’ because of its involvement in cellular inactivation processes (e.g. Ca2 lowering) or in ‘nitrosative stress’ (Ca2 increases). According to our data we would like to propose that the actions of NO have always to be considered in relation to superoxide (+ O2 ) which acts as an antagonist to NO. From a reaction of
* Corresponding author. Tel.: /49-7531-882-287; fax: /497531-884-084. E-mail address: [email protected] (V. Ullrich).
NO and + O2 peroxynitirite is formed which has strong oxidizing properties and can be responsible for pathophysiological effects of NO. Hence, we have studied a possible messenger function of superoxide alone and then its action in the presence of NO. +
2. Superoxide as a messenger Just by scavenging NO superoxide exhibits messenger functions, but recent observations on the regulation of calcineurin (CaN, PP2B) provide evidence that O2 can directly control enzyme activity (Namgaladze et al., 2002). CaN is the only calcium/calmodulin-dependent protein serine/ threonine phosphatase, and it belongs to the class of metallophosphatases containing binuclear metal
0378-4274/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 4 2 7 4 ( 0 2 ) 0 0 4 2 4 - 1
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V. Ullrich et al. / Toxicology Letters 139 (2003) 107 /110
clusters at the active site. Other major protein serine/threonine phosphatases, PP1 and PP2A, as well as purple acid phosphatases, also have similar binuclear metal centers. CaN contains iron and zinc in the binuclear center (King and Huang, 1984), and it was shown that the redox state of iron affects phosphatase activity of CaN and purple acid phosphatases (Davis and Averill, 1982; Yu et al., 1995). First indication of CaN superoxide sensitivity came from the study, which showed protection of CaN activity in brain homogenates against calcium-dependent inactivation by Cu,Zn-SOD (Wang et al., 1996). In our work we demonstrated that superoxide indeed is a very potent CaN inhibiting species, with a potency at least three orders of magnitude over that of H2O2 (Namgaladze et al., 2002). By modifying our CaN purification scheme we succeeded to isolate sufficient quantities of superoxide-sensitive CaN, and investigated the mechanism of inhibition in detail. Particularly, the inhibition was calciumand calmodulin-dependent, indicating that the same conformational rearrangements that cause CaN activation increase its sensitivity to oxidative inactivation. Strikingly, application of NO-donors completely blocked superoxide inhibitory effects. Thus, NO generation in this system again would play an essential regulatory role by antagonizing the action of superoxide. Apparently, neither peroxynitrite nor other species arising from NO/ O2 system are able to inhibit CaN phosphatase activity to the extent seen with superoxide. By analyzing the possible targets of superoxide inhibitory action on the enzyme we obtained sufficient evidence for the critical role of ferrous Fe in the enzyme binuclear center as a superoxide target. Particularly, the enzyme was fully protected against O2 by ascorbate; furthermore, after inactivation it was possible to partly reactivate the enzyme by ascorbate treatment and almost complete re-activation was achieved by a mixture of Fe(II) and ascorbate. Conclusive evidence came from EPR spectroscopic investigation, which showed that the native enzyme form contains mostly ferrous iron, which could be oxidized to the inactive ferric state. These data allow us to suggest the following scheme of CaN inhibition by superoxide (Fig. 1).
According to this scheme, calcium/calmodulindependent activation of the enzyme leads to the opening of the enzyme active site, allowing not only the access of substrates, but also an easier attack of superoxide. The putative m-peroxo-like intermediate should be stabilized by electrostatic interactions with a zinc ion. Peroxide release leaves an inactive Fe(III)/Zn enzyme. Our data indicate that this ferric iron is rather labile and can be easily exchanged for several divalent cations, e.g. manganese, with subsequent enzyme activation. Calcineurin appears to be a crucial cellular target of superoxide, since it is a key regulator of various signaling processes governed by reversible serine/threonine phosphorylation. Of particular importance is its role in neuronal cells, where CaN was shown to participate in processes of synaptic plasticity, memory formation, gene transcription or apoptosis. It is essential that CaN seems to be involved in the pathogenesis of several neurodegenerative diseases, which are accompanied by increased oxidative stress. Thus, CaN activity was shown to be sensitive to the mutations in Cu,Zn-SOD, which occur in familial amyotrophic lateral sclerosis (FALS), a fatal neurodegenerative disease affecting motor neurons (Ferri et al., 2001; Volkel et al., 2001). CaN mRNA appears to be the most elevated mRNA in patients with Alzheimer’s disease (Hata et al., 2001). Similarly elevated is an endogenous regulator of CaN, DSCR1/MCIP1 (Ermak et al., 2001). All these findings point to the link between redox processes and calcineurin activity in neuronal pathophysiology. Superoxide is a weak oxidant unless like in CaN it acquires an electron and then becomes a peroxide. The same activation occurs with NO when peroxynitrite is formed. This peroxide compared to hydrogen peroxide is highly reactive and can oxidize sulfhydryls, methonine or can nitrate tyrosine residues to 3-nitrotyrosine (Beckman and Koppenol, 1996). This is the underlying mechanism for prostacyclin synthase inhibition (Zou et al., 1997), for which we have postulated a role in ischemia/reperfusion (Zou and Bachschmid, 1999) or atherosclerosis (Zou et al., 1999). Considering the low levels of NO and superoxide the resulting PN must act quite specifically if it serves as a
V. Ullrich et al. / Toxicology Letters 139 (2003) 107 /110
109
Fig. 1. Putative mechanism of calcineurin inhibition by superoxide. AI, autoinhibitory domain; CAM, calmodulin.
messenger. We have reported that this can be achieved not only by tyrosine nitration, but also by oxidation of vicinal SH-groups in zinc finger motifs (Daiber et al., 2002). With alcohol dehydrogenase as a model it was shown that Zn2 is released by formation of a disulfide. SIN-1, which generates NO and O2 simultaneously, is an efficient agent for oxidation and Cu,Zn-SOD is almost completely inhibiting. This proves that NO alone has no oxidizing power in contrast to reports in literature. We finally could show that an excess of NO compared to O2 effectively quenches peroxynitrite and generates a nitrosating species, probably N2O3 according to the equation
NO
OON ONO 0 NO2 + NO2 0 N2 O3
This may be a route by which S-nitrosation can occur in vivo. In summary, superoxide seems to have similar messenger functions as NO, but in an antagonistic sense.
References Beckman, J.S., Koppenol, W.H., 1996. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am. J. Physiol. 271, C1424 /C1437. Daiber, A., Frein, D., Namgaladze, D., Ullrich, V., 2002. Oxidation and nitrosation in the nitrogen monoxide/superoxide system. J. Biol. Chem. 277, 11882 /11888.
Davis, J.C., Averill, B.A., 1982. Evidence for a spin-coupled binuclear iron unit at the active site of the purple acid phosphatase from beef spleen. Proc. Natl. Acad. Sci. USA 79, 4623 /4627. Ermak, G., Morgan, T.E., Davies, K.J., 2001. Chronic overexpression of the calcineurin inhibitory gene DSCR1 (Adapt78) is associated with Alzheimer’s disease. J. Biol. Chem. 276, 38787 /38794. Ferri, A., Gabbianelli, R., Casciati, A., Celsi, F., Rotilio, G., Carri, M.T., 2001. Oxidative inactivation of calcineurin by Cu,Zn superoxide dismutase G93A, a mutant typical of familial amyotrophic lateral sclerosis. J. Neurochem. 79, 531 /538. Hata, R., Masumura, M., Akatsu, H., Li, F., Fujita, H., Nagai, Y., Yamamoto, T., Okada, H., Kosaka, K., Sakanaka, M., Sawada, T., 2001. Up-regulation of calcineurin Abeta mRNA in the Alzheimer’s disease brain: assessment by cDNA microarray. Biochem. Biophys. Res. Commun. 284, 310 /316. King, M.M., Huang, C.Y., 1984. The calmodulin-dependent activation and deactivation of the phosphoprotein phosphatase, calcineurin, and the effect of nucleotides, pyrophosphate, and divalent metal ions. Identification of calcineurin as a Zn and Fe metalloenzyme. J. Biol. Chem. 259, 8847 / 8856. Namgaladze, D., Hofer, H.W., Ullrich, V., 2002. Redox control of calcineurin by targeting the binuclear Fe(2/)-Zn(2/) center at the enzyme active site. J. Biol. Chem. 277, 5962 / 5969. Volkel, H., Scholz, M., Link, J., Selzle, M., Werner, P., Tunnemann, R., Jung, G., Ludolph, A.C., Reuter, A., 2001. Superoxide dismutase mutations of familial amyotrophic lateral sclerosis and the oxidative inactivation of calcineurin. FEBS Lett. 503, 201 /205. Wang, X., Culotta, V.C., Klee, C.B., 1996. Superoxide dismutase protects calcineurin from inactivation. Nature (Lond.) 383, 434 /437.
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Yu, L., Haddy, A., Rusnak, F., 1995. Evidence that calcineurin accommodates an active site binuclear metal center. J. Am. Chem. Soc. 117, 10147 /10148. Zou, M.H., Bachschmid, M., 1999. Hypoxia-reoxygenation triggers coronary vasospasm in isolated bovine coronary arteries via tyrosine nitration of prostacyclin synthase. J. Exp. Med. 190, 135 /139.
Zou, M., Martin, C., Ullrich, V., 1997. Tyrosine nitration as a mechanism of selective inactivation of prostacyclin synthase by peroxynitrite. Biol. Chem. 378, 707 /713. Zou, M.H., Leist, M., Ullrich, V., 1999. Selective nitration of prostacyclin synthase and defective vasorelaxation in atherosclerotic bovine coronary arteries. Am. J. Pathol. 154, 1359 /1365.
Short communication
Superoxide as inhibitor of calcineurin and mediator of redox regulation Volker Ullrich *, Dmitry Namgaladze, Daniel Frein Mathematisch-Naturwissenschaftliche Sektion, Fachbereich Biologie, Universita¨t Konstanz, D-78457 Konstanz, Germany
Abstract The concept of NO as a redoxactive messenger has to be broadened by including superoxide as an antagonistic messenger. Superoxide alone was found to inhibit calcineurin by interacting with the FeII /ZnII binuclear site. This links oxidative stress conditions with a Ca-dependent phosphorylation/dephosphorylation cascade. When NO and superoxide are generated at equal fluxes the resulting peroxynitrite can cause tyrosine nitrations (e.g. prostacyclin synthase inhibition) or oxidations of zinc-fingers in proteins, indicating a new messenger function. Finally, if generated in excess, NO can convert peroxynitrite to N2O3 as a nitrosating agent. Thus, the NO/superoxide system provides four different messengers affecting important regulatory pathways. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Nitrogen monoxide/superoxide system; Peroxynitrite; Calcineurin
1. Introduction Nitrogen monoxide (nitric oxide, NO) has important functions as a signaling molecule but no clear-cut concept is available for its physiological effects since it has been described as a ‘double-edged sword’ because of its involvement in cellular inactivation processes (e.g. Ca2 lowering) or in ‘nitrosative stress’ (Ca2 increases). According to our data we would like to propose that the actions of NO have always to be considered in relation to superoxide (+ O2 ) which acts as an antagonist to NO. From a reaction of
* Corresponding author. Tel.: /49-7531-882-287; fax: /497531-884-084. E-mail address: [email protected] (V. Ullrich).
NO and + O2 peroxynitirite is formed which has strong oxidizing properties and can be responsible for pathophysiological effects of NO. Hence, we have studied a possible messenger function of superoxide alone and then its action in the presence of NO. +
2. Superoxide as a messenger Just by scavenging NO superoxide exhibits messenger functions, but recent observations on the regulation of calcineurin (CaN, PP2B) provide evidence that O2 can directly control enzyme activity (Namgaladze et al., 2002). CaN is the only calcium/calmodulin-dependent protein serine/ threonine phosphatase, and it belongs to the class of metallophosphatases containing binuclear metal
0378-4274/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 4 2 7 4 ( 0 2 ) 0 0 4 2 4 - 1
108
V. Ullrich et al. / Toxicology Letters 139 (2003) 107 /110
clusters at the active site. Other major protein serine/threonine phosphatases, PP1 and PP2A, as well as purple acid phosphatases, also have similar binuclear metal centers. CaN contains iron and zinc in the binuclear center (King and Huang, 1984), and it was shown that the redox state of iron affects phosphatase activity of CaN and purple acid phosphatases (Davis and Averill, 1982; Yu et al., 1995). First indication of CaN superoxide sensitivity came from the study, which showed protection of CaN activity in brain homogenates against calcium-dependent inactivation by Cu,Zn-SOD (Wang et al., 1996). In our work we demonstrated that superoxide indeed is a very potent CaN inhibiting species, with a potency at least three orders of magnitude over that of H2O2 (Namgaladze et al., 2002). By modifying our CaN purification scheme we succeeded to isolate sufficient quantities of superoxide-sensitive CaN, and investigated the mechanism of inhibition in detail. Particularly, the inhibition was calciumand calmodulin-dependent, indicating that the same conformational rearrangements that cause CaN activation increase its sensitivity to oxidative inactivation. Strikingly, application of NO-donors completely blocked superoxide inhibitory effects. Thus, NO generation in this system again would play an essential regulatory role by antagonizing the action of superoxide. Apparently, neither peroxynitrite nor other species arising from NO/ O2 system are able to inhibit CaN phosphatase activity to the extent seen with superoxide. By analyzing the possible targets of superoxide inhibitory action on the enzyme we obtained sufficient evidence for the critical role of ferrous Fe in the enzyme binuclear center as a superoxide target. Particularly, the enzyme was fully protected against O2 by ascorbate; furthermore, after inactivation it was possible to partly reactivate the enzyme by ascorbate treatment and almost complete re-activation was achieved by a mixture of Fe(II) and ascorbate. Conclusive evidence came from EPR spectroscopic investigation, which showed that the native enzyme form contains mostly ferrous iron, which could be oxidized to the inactive ferric state. These data allow us to suggest the following scheme of CaN inhibition by superoxide (Fig. 1).
According to this scheme, calcium/calmodulindependent activation of the enzyme leads to the opening of the enzyme active site, allowing not only the access of substrates, but also an easier attack of superoxide. The putative m-peroxo-like intermediate should be stabilized by electrostatic interactions with a zinc ion. Peroxide release leaves an inactive Fe(III)/Zn enzyme. Our data indicate that this ferric iron is rather labile and can be easily exchanged for several divalent cations, e.g. manganese, with subsequent enzyme activation. Calcineurin appears to be a crucial cellular target of superoxide, since it is a key regulator of various signaling processes governed by reversible serine/threonine phosphorylation. Of particular importance is its role in neuronal cells, where CaN was shown to participate in processes of synaptic plasticity, memory formation, gene transcription or apoptosis. It is essential that CaN seems to be involved in the pathogenesis of several neurodegenerative diseases, which are accompanied by increased oxidative stress. Thus, CaN activity was shown to be sensitive to the mutations in Cu,Zn-SOD, which occur in familial amyotrophic lateral sclerosis (FALS), a fatal neurodegenerative disease affecting motor neurons (Ferri et al., 2001; Volkel et al., 2001). CaN mRNA appears to be the most elevated mRNA in patients with Alzheimer’s disease (Hata et al., 2001). Similarly elevated is an endogenous regulator of CaN, DSCR1/MCIP1 (Ermak et al., 2001). All these findings point to the link between redox processes and calcineurin activity in neuronal pathophysiology. Superoxide is a weak oxidant unless like in CaN it acquires an electron and then becomes a peroxide. The same activation occurs with NO when peroxynitrite is formed. This peroxide compared to hydrogen peroxide is highly reactive and can oxidize sulfhydryls, methonine or can nitrate tyrosine residues to 3-nitrotyrosine (Beckman and Koppenol, 1996). This is the underlying mechanism for prostacyclin synthase inhibition (Zou et al., 1997), for which we have postulated a role in ischemia/reperfusion (Zou and Bachschmid, 1999) or atherosclerosis (Zou et al., 1999). Considering the low levels of NO and superoxide the resulting PN must act quite specifically if it serves as a
V. Ullrich et al. / Toxicology Letters 139 (2003) 107 /110
109
Fig. 1. Putative mechanism of calcineurin inhibition by superoxide. AI, autoinhibitory domain; CAM, calmodulin.
messenger. We have reported that this can be achieved not only by tyrosine nitration, but also by oxidation of vicinal SH-groups in zinc finger motifs (Daiber et al., 2002). With alcohol dehydrogenase as a model it was shown that Zn2 is released by formation of a disulfide. SIN-1, which generates NO and O2 simultaneously, is an efficient agent for oxidation and Cu,Zn-SOD is almost completely inhibiting. This proves that NO alone has no oxidizing power in contrast to reports in literature. We finally could show that an excess of NO compared to O2 effectively quenches peroxynitrite and generates a nitrosating species, probably N2O3 according to the equation
NO
OON ONO 0 NO2 + NO2 0 N2 O3
This may be a route by which S-nitrosation can occur in vivo. In summary, superoxide seems to have similar messenger functions as NO, but in an antagonistic sense.
References Beckman, J.S., Koppenol, W.H., 1996. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am. J. Physiol. 271, C1424 /C1437. Daiber, A., Frein, D., Namgaladze, D., Ullrich, V., 2002. Oxidation and nitrosation in the nitrogen monoxide/superoxide system. J. Biol. Chem. 277, 11882 /11888.
Davis, J.C., Averill, B.A., 1982. Evidence for a spin-coupled binuclear iron unit at the active site of the purple acid phosphatase from beef spleen. Proc. Natl. Acad. Sci. USA 79, 4623 /4627. Ermak, G., Morgan, T.E., Davies, K.J., 2001. Chronic overexpression of the calcineurin inhibitory gene DSCR1 (Adapt78) is associated with Alzheimer’s disease. J. Biol. Chem. 276, 38787 /38794. Ferri, A., Gabbianelli, R., Casciati, A., Celsi, F., Rotilio, G., Carri, M.T., 2001. Oxidative inactivation of calcineurin by Cu,Zn superoxide dismutase G93A, a mutant typical of familial amyotrophic lateral sclerosis. J. Neurochem. 79, 531 /538. Hata, R., Masumura, M., Akatsu, H., Li, F., Fujita, H., Nagai, Y., Yamamoto, T., Okada, H., Kosaka, K., Sakanaka, M., Sawada, T., 2001. Up-regulation of calcineurin Abeta mRNA in the Alzheimer’s disease brain: assessment by cDNA microarray. Biochem. Biophys. Res. Commun. 284, 310 /316. King, M.M., Huang, C.Y., 1984. The calmodulin-dependent activation and deactivation of the phosphoprotein phosphatase, calcineurin, and the effect of nucleotides, pyrophosphate, and divalent metal ions. Identification of calcineurin as a Zn and Fe metalloenzyme. J. Biol. Chem. 259, 8847 / 8856. Namgaladze, D., Hofer, H.W., Ullrich, V., 2002. Redox control of calcineurin by targeting the binuclear Fe(2/)-Zn(2/) center at the enzyme active site. J. Biol. Chem. 277, 5962 / 5969. Volkel, H., Scholz, M., Link, J., Selzle, M., Werner, P., Tunnemann, R., Jung, G., Ludolph, A.C., Reuter, A., 2001. Superoxide dismutase mutations of familial amyotrophic lateral sclerosis and the oxidative inactivation of calcineurin. FEBS Lett. 503, 201 /205. Wang, X., Culotta, V.C., Klee, C.B., 1996. Superoxide dismutase protects calcineurin from inactivation. Nature (Lond.) 383, 434 /437.
110
V. Ullrich et al. / Toxicology Letters 139 (2003) 107 /110
Yu, L., Haddy, A., Rusnak, F., 1995. Evidence that calcineurin accommodates an active site binuclear metal center. J. Am. Chem. Soc. 117, 10147 /10148. Zou, M.H., Bachschmid, M., 1999. Hypoxia-reoxygenation triggers coronary vasospasm in isolated bovine coronary arteries via tyrosine nitration of prostacyclin synthase. J. Exp. Med. 190, 135 /139.
Zou, M., Martin, C., Ullrich, V., 1997. Tyrosine nitration as a mechanism of selective inactivation of prostacyclin synthase by peroxynitrite. Biol. Chem. 378, 707 /713. Zou, M.H., Leist, M., Ullrich, V., 1999. Selective nitration of prostacyclin synthase and defective vasorelaxation in atherosclerotic bovine coronary arteries. Am. J. Pathol. 154, 1359 /1365.