Interaction of peroxynitrite with selenoproteins and glutathione peroxidase mimics

Free Radical Biology & Medicine, Vol. 28, No. 10, pp. 1451–1455, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/00/$–see front matter

PII S0891-5849(00)00253-7

Forum: Therapeutic Applications of Reactive Oxygen and Nitrogen Species in Human Disease INTERACTION OF PEROXYNITRITE WITH SELENOPROTEINS AND GLUTATHIONE PEROXIDASE MIMICS HELMUT SIES

and

GAVIN E. ARTEEL

Institut fu¨r Physiologische Chemie I, Heinrich-Heine-Universita¨t Du¨sseldorf, Du¨sseldorf, Germany (Received 4 January 2000; Accepted 20 January 2000)

Abstract—Peroxynitrite is an oxidant generated under inflammatory conditions, acting in defense against invading microorganisms. There is a need for protection of the organism from damage inflicted by peroxynitrite. Seleniumcontaining compounds, notably ebselen, have a high second-order reaction rate constant (approx. 2 ⫻ 106 M⫺1 s⫺1), which makes them candidates for efficient protection. This applies also for selenium in proteins, occurring as selenocysteine or selenomethionine residues. Glutathione peroxidases, thioredoxin reductase, and selenoprotein P have been shown to play a potential role in protection against peroxynitrite. Tellurium-containing compounds also react with peroxynitrite. © 2000 Elsevier Science Inc. Keywords—Ebselen, GSH peroxidase, Peroxynitrite, Plasma, Oxidative stress, Free radicals

INTRODUCTION

cept this reactive oxygen/nitrogen species so that potentially sensitive biological targets cannot be reached and damage is prevented [1]. Such a strategy requires that the intercepting molecule is available at the corresponding site near the target to be protected, or near the source of peroxynitrite generation. Further, the reaction product formed between peroxynitrite and the intercepting molecule should be recyclable in order to permit the maintenance of a defense line, that is, a “catalytic” protection would be desirable rather than a “one-shot-only” strategy, in which stoichiometric amounts of a protective molecule would be utilized in case the reaction product cannot be repaired. For assessing the detoxifying capacity of a given compound with a prooxidant, it is useful to consider the rate constant of this reaction. Table 1 lists the rate constants for some selenium-containing compounds and proteins as well as the concentration required to inhibit the oxidation of dihydrorhodamine 123 by peroxynitrite by 50%. Organoselenium compounds fulfill such requirements. In 1996, it was found that the organoselenium compound ebselen reacted in a rapid reaction with peroxynitrite [2], the second-order rate constant being 2.0 ⫻ 106 M⫺1s⫺1 [3]. With ebselen being known as a glutathione peroxidase mimic [4,5], interest turned to biologically occurring organoselenium compounds in proteins on one hand, and to other synthetic organoselenium and organotellurium compounds on the other. The present forum article focuses on the interactions of peroxynitrite,

The defenses against peroxynitrite are multilayered. In protection against peroxynitrite, one strategy is to interHelmut Sies, M.D., Ph.D.(hon.), is Professor and Chairman, Department of Physiological Chemistry I, at the Faculty of Medicine, Heinrich-Heine University at Du¨sseldorf, Germany, since 1979. After studying medicine at Tu¨bingen, Paris and Munich (M.D., 1967), he received his Habilitation for Physiological Chemistry and Physical Biochemistry at the University of Munich in 1972, and an Honorary Ph.D. from the University of Buenos Aires, Argentina, in 1996. He worked with Britton Chance, Johnson Research Foundation (Philadelphia, 1969 – 1970) and was Visiting Professor at the University of California at Berkeley , Department of Biochemistry (Bruce Ames, 1984 –1985) and Department of Molecular and Cell Biology as Miller Visiting Professor (Lester Packer, 1992), and at the Heart Research Institute, Sydney (Roland Stocker, 1993). He is President of the Society for Free Radical Research (International) 1998 –2000. His research interests in biological oxidations include oxidative stress, oxidants, and antioxidants (glutathione, tocopherols, carotenoids, flavonoids, peroxynitrite and selenium). G.E. Arteel obtained his B.S. (1993) in Pharmacology and Toxicology at the School of Pharmacy at the University of Wisconsin-Madison. His Ph.D. (1997) in Toxicology was at the University of North Carolina-Chapel Hill, under Dr. James Raleigh and Dr. Ronald G. Thurman. There, he focused on mechanisms by which alcohol consumption damages the liver, concentrating on the involvement of hypoxia, subsequent reoxygenation, and reactive species formation. Subsequently, he was an Alexander von Humboldt post-doctoral fellow under Prof. Dr. Helmut Sies in Du¨sseldorf, Germany (1998 –1999), investigating biochemical protection against peroxynitrite. He is currently a post-doctoral fellow of the Bowles Center for Alcohol Studies at the University of North Carolina-Chapel Hill. Address correspondence to: Dr. Helmut Sies, Institut fu¨r Physiologische Chemie I, Universita¨t Du¨sseldorf, Postfach 101007, D-40001Du¨sseldorf, Germany; Tel: ⫹49 (211) 811-2707; Fax: ⫹49 (211) 811-3029; E-Mail: [email protected]. 1451

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H. SIES and G. E. ARTEEL Table 1. Interception of Peroxynitrite by Selenium-containing Compounds and Proteins b

Rate constant

Dihydrorhodamine-123 oxidation half-maximal inhibitory concentration

(M⫺1s⫺1)

(␮M)

— 8.0 ⫻ 106 [20] — 7.4 ⫻ 105 [20] 5.6 ⫻ 103 [37]

0.05 [17] 0.2 [19] 0.1 [19] — 7 [19]

2.0 ⫻ 106 [3] — 1.2 ⫻ 104 [38] — 2.4 ⫻ 103 [16] 1.8 ⫻ 102 [39] — — — 5.8 ⫻ 102 [40] —

0.15 [17] 15 [17] 0.8 [19] 100 [19] 0.3 [17] 20 [17] 2.5 [17] ⬎103 [17] ⬎104 [17] 12 [19] 0.08 [36]

a

Compound Proteins PHGPx Glutathione peroxidase (GPx) Carboxymethylated GPx Oxidized GPx Albumin Small Molecules Ebselen Ebsulfur 2-(Methylseleno)benzanilide Ebselen selenoxide Selenomethionine Methionine Selenocystine Cystine Sodium selenite Glutathione Bis[4-aminophenyl]telluride a b

Second-order rate constants are for pH 7.4, 25°C. Dihydrorhodamine 123 (0.5 ␮M), DTPA (0.1 mM), and peroxynitrite (0.1 ␮M).

with the following subtopics: (i) a brief presentation of ebselen, (ii) selenocysteine and selenomethionine, (iii) selenoproteins, and (iv) organotellurium compounds.

oredoxin reductase can also reduce ebselen selenoxide at the expense of NADPH ([13]; see below). Thus, ebselen can act both as a catalyst reducing hydroperoxides, and as a catalyst reducing peroxynitrite (see [14]).

EBSELEN

This compound has been extensively studied as a glutathione peroxidase mimic (see [6] for review). Ebselen exhibits anti-inflammatory actions in a number of experimental models [6 – 8]. The compound has been developed for clinical use (Phase III), as recently reviewed [9]. The indications addressed were acute ischemic stroke [10], delayed neurological deficits after subarachnoid hemorrhage [11], and acute middle cerebral artery occlusion [12]. These clinical conditions are associated with an inflammatory component, and it would appear reasonable to assume that potential beneficial effects may be linked to the known anti-inflammatory activity of ebselen. One major aspect in this regard has been the activity of the compound as a glutathione peroxidase mimic [4]. It was of considerable interest to note that ebselen also reacts with peroxynitrite, a potent inflammatory mediator [2,3]. The compound acts catalytically in reducing peroxynitrite to nitrite in a first step, with the reaction product being the corresponding selenoxide, followed by the reduction of this selenoxide back to ebselen in two consecutive one-electron reduction steps via the selenodisulfide, utilizing reducing equivalents in the form of glutathione. Further, the mammalian selenoprotein thi-

SELENOCYSTEINE AND SELENOMETHIONINE

The major selenium-containing aminoacids in the body are selenocysteine and selenomethionine. Selenocysteine is incorporated into a number of selenoproteins (see below) by a specific insertion machinery; selenomethionine is incorporated into protein at random in place of methionine [15]. The reactivity of peroxynitrite is much higher for selenomethionine than methionine, as evidenced by a higher second-order rate constant [16], and by more efficient protection against oxidation and nitration reactions [17]. Likewise, selenocystine was more effective in protection assays than cystine [17]. The reduction of the selenoxide formed upon oxidation at selenocysteine residues or selenomethionine in protein would occur by reducing systems which may or may not be specific. It was found that glutathione, but also dihydrolipoic acid or ␤-mercaptoethanol can reduce the selenoxide of selenocysteine in proteins [18]. Interestingly, the reduction of the selenoxide of selenomethionine by glutathione was quite rapid [18]. However, recent work suggested that albumin from selenium-supplemented individuals did not apparently protect peroxynitrite relative to albumin isolated from individuals prior to selenium supplementation [15], although sel-

Peroxynitrite and selenoproteins

enomethionine levels in albumin were higher after supplementation. However, these experiments were performed using bolus addition of relatively high concentrations of peroxynitrite [15], which could overwhelm the protective potential of selenomethionine residues. SELENOPROTEINS

Similar to organoselenium compounds, selenoproteins may fulfill the above-mentioned requirements as efficient interceptors of peroxynitrite.

Glutathione peroxidase The selenocysteine-containing glutathione peroxidase (GPx) can act as a peroxynitrite reductase, preventing oxidation and nitration reactions caused by peroxynitrite [19]. Glutathione peroxidase reduces peroxynitrite to nitrite using GSH in a catalytic reaction, similar to that described above for ebselen. Increases in nitrite during exposure to peroxynitrite were observed with GPx [19], indicating two-electron reduction of peroxynitrite; however, the nitrite yield was less than complete (⬃50%). The second-order rate constant for the reaction of glutathione peroxidase (tetrameric) with peroxynitrite is 8.0 ⫻ 106 M⫺1 s⫺1 [20]. While there is no net loss of GPx activity when GPx is maintained in the reduced state by supplying reductants [19,20], GPx is inactivated in the absence of GSH [21] or upon exposure to nitric oxide donors [22]. Comparing the second-order rate constants with their respective intracellular concentrations, it is likely that GPx outcompetes thiols for the direct reaction with peroxynitrite. It has been shown that the rate of H2O2 reduction by glutathione peroxidase is the same as the as second-order rate constant of the interaction of GPx with H2O2, since the rate of the reduction of oxidized GPx is not rate-limiting in the presence of physiological (mM) concentrations of GSH [23]. These observations will also apply to the reaction of GPx with peroxynitrite. For homogeneous systems, multiplication of the concentration of a given compound with the corresponding second-order rate constant for the reaction with peroxynitrite yields the rate of disappearance of peroxynitrite; the rate of peroxynitrite reduction by GPx is estimated to be 16 s⫺1 (see ref [1]). Increasing the level of selenoproteins (e.g., GPx 14-fold) by selenium supplementation attenuated mitogen-activated protein kinase (p38, JNK1/2 and ERK1/2) activation by peroxynitrite in cultured WB-F344 rat liver cells [24]. Thus, the reaction of GPx with peroxynitrite is considered a biologically efficient detoxication pathway in vivo. In view of the recently reported evidence for peroxynitrite as a

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signaling molecule in flow-dependent activation of JNK [25], the GPx reaction would also be modulatory in some circumstances. Selenoprotein P Selenoprotein P in human plasma also protects against peroxynitrite [26], suggesting that it may serve as a protectant against peroxynitrite in human blood. The heparin-binding domains of selenoprotein P enable surface coating of cellular membranes (e.g., endothelial cells [27,28]). This effect could serve two purposes in vivo: (i) as a means of concentrating selenoprotein P on cell surfaces for targeted defense against oxidants; (ii) reducing equivalents could be transferred to bound oxidized selenoprotein P from the cell layer, allowing for maintenance of a defense line. Recent work with surface plasmon resonance has indicated that heparin has two binding sites for selenoprotein P, one with a binding constant in the low nM range and the other in the mid nM range [46]. Thioredoxin reductase Thioredoxin reductase, coupled with thioredoxin and NADPH, is an efficient general protein disulfide reductase (see [29] for review). Mammalian thioredoxin reductase is a selenoprotein [30] and has a much broader substrate specificity than its Escherichia coli counterpart, including a number of organoselenium compounds (e.g., selenocystine [31]). Mammalian thioredoxin reductase can function in the reduction of peroxynitrite by selenocysteine or ebselen [13], maintaining these compounds in a catalytic cycle at the expense of NADPH. Upon administration, extracellular ebselen is present in human plasma as an albumin complex [32]. Thioredoxin reductase, coupled with thioredoxin and NADPH, can also reduce the selenenyl sulfide complex of BSA-ebselen and release free ebselen [13]. ORGANOTELLURIUM COMPOUNDS

Organotellurium compounds also protect against oxidation and nitration reactions caused by peroxynitrite [33,34]. Bis[4-aminophenyl] telluride protects against peroxynitrite-mediated oxidation of dihydrorhodamine 123 more efficiently than its selenium analogue or ebselen [33]. In general, organotellurium compounds tend to be more nucleophilic than their corresponding selenium and sulfur derivatives [35]. The order of reactivity (telluride⬎selenide⬎sulfide) correlates with oxidation potentials and is also in accord with the decreasing electronegativity of the elements as one traverses the

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H. SIES and G. E. ARTEEL

group of chalcogens. Recently, similar experiments were performed with water-soluble organotellurium compounds [36]. It is possible that substituted organotellurium compounds, like those tested, may avoid potentially toxic leakage of tellurium in vivo.

[6] [7]

[8] CONCLUDING REMARKS

[9]

Small organoselenium and organotellurium compounds, selenoaminoacids, and selenoproteins have been shown to protect efficiently against peroxynitrite. In general, the first step is the reduction of peroxynitrite to nitrite, and the reaction can be maintained at the expense of reducing equivalents, forming a catalytic cycle. Given this, bolus addition of high concentrations of peroxynitrite may confound experimental results. It is recommended instead that peroxynitrite is infused to maintain a low, steady-state concentration over the course of time in vitro in the cuvette [19], or in cell culture [24], similar to levels of peroxynitrite production in vivo. It should be mentioned that under pathophysiological conditions other reactivities of these compounds can play an important role as well, which may or may not be related to the activity as GPx mimic. GPx mimics have been described as in vivo immune response modifiers [41]. For example, ICAM-1 and VCAM-1 expression induced by TNF-␣ were found to be inhibited [42], as were TNF-␣- and neutrophil-induced endothelial alterations [43] and TNF-mediated apoptosis [44]. In recent work on the inhibition of prostacyclin synthase by peroxynitrite, it was suggested that ebselen in cellular systems is present as thiol adducts and thus loses its high reactivity towards peroxynitrite [45]; this, however, would not apply to selenocysteines or selenomethionines in proteins.

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20] Acknowledgements — Support by the Deutsche Forschungsgemeinschaft, SFB 503, Project B1, and by the National Foundation for Cancer Research, Bethesda, MD, is gratefully acknowledged. G.E. Arteel is a Research Fellow of the Alexander von Humboldt Foundation, Bonn, Germany.

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