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W1991821808 abstract "Macrophage migration inhibitory factor (MIF) is an important mediator that plays a central role in the control of the host immune and inflammatory response. To investigate the molecular mechanism of MIF action, we have used the yeast two-hybrid system and identified PAG, a thiol-specific antioxidant protein, as an interacting partner of MIF. Association of MIF with PAG was found in 293T cells transiently expressing MIF and PAG. The use of PAG mutants (C52S, C71S, and C173S) revealed that this association was significantly affected by C173S, but not C52S and C71S, indicating that a disulfide involving Cys173 of PAG is responsible for the formation of MIF·PAG complex. In addition, the interaction was highly dependent on the reducing conditions such as dithiothreitol or β-mercaptoethanol but not in the presence of H2O2. Analysis of the activities of the interacting proteins showed that the d-dopachrome tautomerase activity of MIF was decreased in a dose-dependent manner by coexpression of wild-type PAG, C52S, and C71S, whereas C173S was almost ineffective, suggesting that the direct interaction may be involved in the control ofd-dopachrome tautomerase activity of MIF. Moreover, MIF has been shown to bind to PAG and it also inhibits the antioxidant activity of PAG. Macrophage migration inhibitory factor (MIF) is an important mediator that plays a central role in the control of the host immune and inflammatory response. To investigate the molecular mechanism of MIF action, we have used the yeast two-hybrid system and identified PAG, a thiol-specific antioxidant protein, as an interacting partner of MIF. Association of MIF with PAG was found in 293T cells transiently expressing MIF and PAG. The use of PAG mutants (C52S, C71S, and C173S) revealed that this association was significantly affected by C173S, but not C52S and C71S, indicating that a disulfide involving Cys173 of PAG is responsible for the formation of MIF·PAG complex. In addition, the interaction was highly dependent on the reducing conditions such as dithiothreitol or β-mercaptoethanol but not in the presence of H2O2. Analysis of the activities of the interacting proteins showed that the d-dopachrome tautomerase activity of MIF was decreased in a dose-dependent manner by coexpression of wild-type PAG, C52S, and C71S, whereas C173S was almost ineffective, suggesting that the direct interaction may be involved in the control ofd-dopachrome tautomerase activity of MIF. Moreover, MIF has been shown to bind to PAG and it also inhibits the antioxidant activity of PAG. macrophage migration inhibitory factor thiol-specific antioxidant peroxiredoxin polyacrylamide gel electrophoresis proliferation-associated gene glutathioneS-transferase dithiothreitol cytomegalovirus glutamine synthetase Macrophage migration inhibitory factor (MIF)1 is a cytokine that plays an important role in the regulation of host immune and inflammatory response (1Bernhagen J. Calandra T. Bucala R. J. Mol. Med. 1998; 76: 151-161Crossref PubMed Scopus (155) Google Scholar, 2Bucala R. Cytokine Growth Factor Rev. 1996; 7: 19-24Crossref PubMed Scopus (63) Google Scholar, 3Calandra T. Bucala R. Crit. Rev. Immunol. 1997; 17: 77-88Crossref PubMed Google Scholar, 4Metz C. Bucala R. Adv. Immunol. 1997; 66: 197-223Crossref PubMed Google Scholar, 5Calandra T. Bernhagen J. Mitchell R.A. Bucala R. J. Exp. Med. 1994; 179: 1902-1985Crossref Scopus (882) Google Scholar, 6Calandra T. Bernhagen J. Metz C.N. Spiegel L.A. Bacher M. Donnelly T. Cerami A. Bucala R. Nature. 1995; 377: 68-71Crossref PubMed Scopus (1045) Google Scholar). MIF is different from other cytokines in that it is found preformed in MIF-expressing cells (7Bernhagen J. Mitchell R.A. Calandra T. Voelter W. Cerami A. Bucala R. Biochemistry. 1994; 33: 14144-14155Crossref PubMed Scopus (374) Google Scholar, 8Liu Y.C. Nakano T. Elly C. Ishizaka K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11227-11231Crossref PubMed Scopus (26) Google Scholar). In addition, MIF has been proposed to catalyze chemical reactions. Structural studies of MIF have led to the suggestion that MIF bears a close architectural similarity to microbial enzymes such as 5-carboxymethyl-2-hydroxymuconate, 4-oxalocrotonate tautomerase, and chorismate mutase, even though these proteins share little homology in the amino acid sequence (9Chook Y.M. Gray J.V. Ke H. Lipscomb W.N. J. Mol. Biol. 1994; 240: 476-500Crossref PubMed Scopus (156) Google Scholar, 10Subramanya H.S. Roper D.I. Dauter D. Dodson E.J. Davies G.J. Wilson K.S. Wigley D.B. Biochemistry. 1996; 35: 792-802Crossref PubMed Scopus (139) Google Scholar, 11Suzuki M. Sugimoto H. Nakagawa A. Tanaka I. Nishihira J. Sakai M. Nat. Struct. Biol. 1996; 3: 259-266Crossref PubMed Scopus (187) Google Scholar, 12Stivers J.T. Abeygunawardana C. Mildvan A.S. Hajipour G. Whitman C.P. Chen L.H. Biochemistry. 1996; 35: 803-813Crossref PubMed Scopus (84) Google Scholar). On the other hand, based on the amino acid sequence homology and structural similarity of MIF withd-dopachrome tautomerase, which convertsd-dopachrome methyl ester to 5,6-dihydroxyindole-2-carboxymethylester, a d-dopachrome tautomerase activity also has been proposed for MIF (13Rosengren E. Bucala R. Arnan P. Jacobsson L. Odh G. Metz C.N. Rorsman H. Mol. Med. 1996; 2: 143-149Crossref PubMed Google Scholar, 14Zhang M. Aman P. Grubb A. Panagopoulos I. Hindemith A. Rosengren E. Rorsman H. FEBS Lett. 1995; 373: 203-206Crossref PubMed Scopus (45) Google Scholar). However, the physiological significance is currently unclear, because natural substrates of MIF have not yet been found. MIF was demonstrated to catalyze the keto-enol isomerization of bothp-hydroxyphenylpyruvate and phenylpyruvate, a hydroxyphenylpyruvate tautomerase activity (15Rosengren E. Aman P. Thelin S. Hansson C. Ahlfors S. Bjork P. Jacobsson L. Rorsman H. FEBS Lett. 1997; 417: 85-88Crossref PubMed Scopus (207) Google Scholar). More recently, MIF has been reported to possess a thiol-protein oxidoreductase activity (16Kleemann R. Kapurniotu A. Frank R.W. Gessner A. Mischke R. Flieger O. Juttner S. Brunner H. Bernhagen J. J. Mol. Biol. 1998; 280: 85-102Crossref PubMed Scopus (267) Google Scholar), which involves the reduction of insulin and 2- hydroxyethyldisulfide.Several lines of evidence suggest that the intracellular redox regulation might be involved in a variety of cellular functions, including cell proliferation, differentiation, tumor promotion, and apoptosis (17Schreck R. Rieber P. Baeuerle P.A. EMBO J. 1991; 10: 2247-2258Crossref PubMed Scopus (3396) Google Scholar, 18Kalebic T. Kinter A. Poli G. Anderson M.E. Meister A. Fauci A.S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 986-990Crossref PubMed Scopus (319) Google Scholar). Antioxidants govern the intracellular redox status. PAG, a known thiol-specific antioxidant, is a member of the peroxiredoxin (Prx) protein family, which was previously referred to as the alkyl hydroperoxide reductase/thiol-specific antioxidant family and is constitutively expressed in most human tissues, but its expression is higher in organs having a higher level of proliferation (19Prosperi M.-T. Ferbus D. Karczinski I. Goubin G. J. Biol. Chem. 1993; 268: 11050-11056Abstract Full Text PDF PubMed Google Scholar, 20Chae H.Z. Robison K. Poole L.B. Church G. Storz G. Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7017-7021Crossref PubMed Scopus (697) Google Scholar). Many Prx proteins are highly conserved in a wide variety of mammalian species such as human, mouse, and bovine, suggesting a biological importance of this type of enzyme (20Chae H.Z. Robison K. Poole L.B. Church G. Storz G. Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7017-7021Crossref PubMed Scopus (697) Google Scholar, 21Chae H.Z. Chung S.J. Rhee S.G. J. Biol. Chem. 1994; 269: 27670-27678Abstract Full Text PDF PubMed Google Scholar, 22Rhee S.G. Chae H.Z. Biofactors. 1994; 4: 177-180PubMed Google Scholar). Most Prx family members contain two conserved cysteines that correspond to Cys47 and Cys170 of yeast thioredoxin peroxidase. In yeast, both Cys47 and Cys170were shown to be necessary for the formation of intermolecular disulfide bonds, and Cys47, but not Cys170, was the primary site of oxidation by H2O2 (23Chae H.Z. Uhm T.B. Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7022-7026Crossref PubMed Scopus (280) Google Scholar).Here we show that PAG binds specifically to MIF in vivo, and we found that this interaction is dependent on the redox status in that the interaction was significantly affected under reducing conditions. Binding of PAG to MIF can repress the d-dopachrome tautomerase activity of MIF. Moreover, this binding resulted in the suppression of the antioxidant activity of PAG.DISCUSSIONThe present study demonstrates that MIF interacts with PAGin vivo, and that the MIF·PAG interaction is dependent on the redox status and the cysteine residues of PAG. In addition, we found that both d-dopachrome tautomerase activity of MIF and antioxidant activity of PAG were negatively regulated by direct association of MIF with PAG.MIF has been proposed as an unusual cytokine containing a combined function both as a conventional cytokine and as an enzyme (1Bernhagen J. Calandra T. Bucala R. J. Mol. Med. 1998; 76: 151-161Crossref PubMed Scopus (155) Google Scholar, 13Rosengren E. Bucala R. Arnan P. Jacobsson L. Odh G. Metz C.N. Rorsman H. Mol. Med. 1996; 2: 143-149Crossref PubMed Google Scholar, 16Kleemann R. Kapurniotu A. Frank R.W. Gessner A. Mischke R. Flieger O. Juttner S. Brunner H. Bernhagen J. J. Mol. Biol. 1998; 280: 85-102Crossref PubMed Scopus (267) Google Scholar). Recently, several studies have reported a variety of enzymatic functions for MIF, including d-dopachrome tautomerase (13Rosengren E. Bucala R. Arnan P. Jacobsson L. Odh G. Metz C.N. Rorsman H. Mol. Med. 1996; 2: 143-149Crossref PubMed Google Scholar, 14Zhang M. Aman P. Grubb A. Panagopoulos I. Hindemith A. Rosengren E. Rorsman H. FEBS Lett. 1995; 373: 203-206Crossref PubMed Scopus (45) Google Scholar), phenylpyruvate tautomerase (15Rosengren E. Aman P. Thelin S. Hansson C. Ahlfors S. Bjork P. Jacobsson L. Rorsman H. FEBS Lett. 1997; 417: 85-88Crossref PubMed Scopus (207) Google Scholar), and a thiol protein oxidoreductase (16Kleemann R. Kapurniotu A. Frank R.W. Gessner A. Mischke R. Flieger O. Juttner S. Brunner H. Bernhagen J. J. Mol. Biol. 1998; 280: 85-102Crossref PubMed Scopus (267) Google Scholar). d-dopachrome tautomerase was discovered during the study of melanin biosynthesis (13Rosengren E. Bucala R. Arnan P. Jacobsson L. Odh G. Metz C.N. Rorsman H. Mol. Med. 1996; 2: 143-149Crossref PubMed Google Scholar). This enzyme was found to have an amino acid sequence that is highly homologous with that of MIF. It has previously been shown that the N-terminal proline of MIF is required for the d-dopachrome tautomerase and phenylpyruvate tautomerase activities (32Swope M. Sun H.W. Blake P.R. Lolis E. EMBO J. 1998; 17: 3534-3541Crossref PubMed Scopus (180) Google Scholar, 33Lubetsky J.B. Swope M. Dealwis C. Blake P. Lolis E. Biochemistry. 1999; 38: 7346-7354Crossref PubMed Scopus (130) Google Scholar, 34Stamps S.L. Fitzgerald M.C. Whitman C.P. Biochemistry. 1998; 37: 10195-10202Crossref PubMed Scopus (71) Google Scholar). However, based on the observed sequence homology with known thiol-protein oxidoreductases (35Puig A. Lyles M.M. Noiva R. Gilbert H.F. J. Biol. Chem. 1994; 269: 19128-19135Abstract Full Text PDF PubMed Google Scholar), it is conceivable that MIF may exhibit a cysteine-dependent enzymatic oxidoreductase activity and that this activity is dependent on the redox-active conserved sequence motif (Cys-X-X-Cys). Analysis of the amino acid sequence of MIF revealed that there was a conserved sequence motif consisting of Cys57-Ala-Leu-Cys60 that was found to be present in the catalytic center of thiol-protein oxidoreductases such as thioredoxin, protein disulfide isomerase, and glutaredoxin. Consistent with this fact, recently a critical role of the conserved cysteine sequence motif (Cys-X-X-Cys) in the oxidoreductase and macrophage-activating activities of MIF was reported (16Kleemann R. Kapurniotu A. Frank R.W. Gessner A. Mischke R. Flieger O. Juttner S. Brunner H. Bernhagen J. J. Mol. Biol. 1998; 280: 85-102Crossref PubMed Scopus (267) Google Scholar). The existence of an intramolecular disulfide bridge was also demonstrated from studies of the conserved cysteine sequence motif, even though previous studies had shown that neither recombinant E. coli-derived MIF nor native MIF from natural cell sources contained an intermolecular disulfide structure (7Bernhagen J. Mitchell R.A. Calandra T. Voelter W. Cerami A. Bucala R. Biochemistry. 1994; 33: 14144-14155Crossref PubMed Scopus (374) Google Scholar, 16Kleemann R. Kapurniotu A. Frank R.W. Gessner A. Mischke R. Flieger O. Juttner S. Brunner H. Bernhagen J. J. Mol. Biol. 1998; 280: 85-102Crossref PubMed Scopus (267) Google Scholar, 36Wistow G.J. Shaughnessy M.P. Lee D.C. Hodin J. Zelenka P.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1272-1275Crossref PubMed Scopus (181) Google Scholar). In addition, MIF was crystallized as a trimer of three identical subunits (11Suzuki M. Sugimoto H. Nakagawa A. Tanaka I. Nishihira J. Sakai M. Nat. Struct. Biol. 1996; 3: 259-266Crossref PubMed Scopus (187) Google Scholar, 28Kato Y. Muto T. Tomura T. Tsumura H. Watarai H. Mikayama T. Ishizaka K. Kuroki R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3007-3010Crossref PubMed Scopus (43) Google Scholar, 29Sun H.W. Bernhagen J. Bucala R. Lolis E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5191-5196Crossref PubMed Scopus (287) Google Scholar). This raises the possibility that MIF may heterodimerize with other cellular proteins in addition to its ability to homodimerize through disulfide linkages between cysteine residues, and that redox regulation may be also involved in the control of this association. To address this question, we sought to identify cellular target proteins that directly associate with MIF. In this study, we report isolation of PAG as an MIF-interacting protein. We performed the coprecipitation experiments of transiently expressed MIF and wild-type PAG, as well as PAG mutants, and demonstrated that MIF associates with wild-type PAG and PAG mutants tested, except for C173S, in mammalian cells (Fig. 2), suggesting that the conserved Cys173 of PAG plays a pivotal role in the formation of intermolecular disulfide linkages between MIF and PAG. However, we cannot rule out the other possibility that the other conserved Cys52 is also involved in the intermolecular disulfide linkages, because we are able to detect a significant decrease in the association of MIF with C52S, although to a somewhat lesser extent, by coprecipitation studies (Fig. 2). To gain more insight into the roles of cysteine residues in the MIF·PAG association, we examined the in vivo binding of MIF and PAG under various redox conditions. As shown in Fig. 3, upon reducing conditions the association was remarkably decreased in a dose-dependent manner, suggesting that the in vivo association of MIF and PAG may be mediated through disulfide linkages of cysteine residues. Recent studies have suggested that MIF and thiol antioxidants could be bridged through inter-chain disulfides (24Jin D.-Y. Chae H.Z. Rhee S.G. Jeang K.-T. J. Biol. Chem. 1997; 272: 30952-30961Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar, 37Jaschke A. Mi H. Tropschug M. J. Mol. Biol. 1998; 277: 763-769Crossref PubMed Scopus (57) Google Scholar, 38Swope M.D. Sun H.-W. Klockow B. Blake P. Lolis E. J. Biol. Chem. 1998; 273: 14877-14884Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). It is necessary to determine whether the conserved cysteine residues of PAG could influence homo- and heterodimerization and whether this complex formation contributes toward the regulation of functional activities of interacting partners.To understand how the interaction contributes to the activity of the respective proteins, the examination of how MIF or PAG binding modulates the interacting protein function seems to be important. As shown in this report, it can be concluded that PAG is a negative regulator of MIF activity. Moreover, we confirmed this observation with the PAG mutants such as C52S, C71S, and C173S. From these mutation experiments, all mutants, except for C173S, resulted in a decrease ofd-dopachrome tautomerase activity of MIF (Fig. 4 and data not shown). These findings can be explained either by a physical binding of PAG to MIF or by an enzymatic function of PAG. However, the fact that C52S mutant, but not C173S, indeed decreases the MIF activity, together with our observation that both Cys52 and Cys173 are equally critical for the antioxidant activity of PAG (data not shown), does not favor the second model describing the importance of the enzymatic function of PAG in the regulation of MIF activity. In addition, it has recently been shown that several MIF residues near the N-terminal proline are perturbed upon addition ofS-hexylglutathione and affected byp-hydroxyphenylpyruvate, a substrate for the phenylpyruvate tautomerase activity of MIF (33Lubetsky J.B. Swope M. Dealwis C. Blake P. Lolis E. Biochemistry. 1999; 38: 7346-7354Crossref PubMed Scopus (130) Google Scholar, 38Swope M.D. Sun H.-W. Klockow B. Blake P. Lolis E. J. Biol. Chem. 1998; 273: 14877-14884Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Because our studies reported here demonstrated that PAG could inhibit the d-dopachrome tautomerase activity of MIF, it is tempting to speculate that this effect is mediated through the perturbation of the residues surrounding the N-terminal proline on the three-dimensional structure of MIF by direct binding of PAG. Based on our observed results, we imagine that rather than direct contact with the residues surrounding the N-terminal proline, the conformational effect that is caused by intermolecular disulfide linkages between cysteine residues of PAG and MIF likely plays a role that is important in the regulation of MIFd-dopachrome tautomerase activity, probably by structural changes within the region near the N-terminal proline.A recent study (30Kleemann R. Mischke R. Kapurniotu A. Brunner H. Bernhagen J. FEBS Lett. 1998; 430: 191-196Crossref PubMed Scopus (61) Google Scholar) demonstrated that Aop1, a human thioredoxin peroxidase, and cyclophilin18 do not only interact physically, but binding of cyclophilin18 also stimulates the antioxidant activity of Aop1, suggesting an important role for the direct interaction in the regulation of the antioxidant activity of thioredoxin peroxidases. To test a possible influence of binding of MIF on the enzymatic activity of PAG, we used a similar approach. As shown in Fig. 5, a dramatic decrease was observed in the antioxidant activity of PAG when increasing amounts of MIF were added to the PAG. This also indicates that MIF negatively regulates the PAG activity. In this regard, the mechanism of interaction of MIF and PAG will be the interest of future study. Further understanding of the mechanism of this interaction will result from the identification of other interacting proteins associated with MIF or PAG and detailed analyses of the binding region in the MIF·PAG interaction. Macrophage migration inhibitory factor (MIF)1 is a cytokine that plays an important role in the regulation of host immune and inflammatory response (1Bernhagen J. Calandra T. Bucala R. J. Mol. Med. 1998; 76: 151-161Crossref PubMed Scopus (155) Google Scholar, 2Bucala R. Cytokine Growth Factor Rev. 1996; 7: 19-24Crossref PubMed Scopus (63) Google Scholar, 3Calandra T. Bucala R. Crit. Rev. Immunol. 1997; 17: 77-88Crossref PubMed Google Scholar, 4Metz C. Bucala R. Adv. Immunol. 1997; 66: 197-223Crossref PubMed Google Scholar, 5Calandra T. Bernhagen J. Mitchell R.A. Bucala R. J. Exp. Med. 1994; 179: 1902-1985Crossref Scopus (882) Google Scholar, 6Calandra T. Bernhagen J. Metz C.N. Spiegel L.A. Bacher M. Donnelly T. Cerami A. Bucala R. Nature. 1995; 377: 68-71Crossref PubMed Scopus (1045) Google Scholar). MIF is different from other cytokines in that it is found preformed in MIF-expressing cells (7Bernhagen J. Mitchell R.A. Calandra T. Voelter W. Cerami A. Bucala R. Biochemistry. 1994; 33: 14144-14155Crossref PubMed Scopus (374) Google Scholar, 8Liu Y.C. Nakano T. Elly C. Ishizaka K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11227-11231Crossref PubMed Scopus (26) Google Scholar). In addition, MIF has been proposed to catalyze chemical reactions. Structural studies of MIF have led to the suggestion that MIF bears a close architectural similarity to microbial enzymes such as 5-carboxymethyl-2-hydroxymuconate, 4-oxalocrotonate tautomerase, and chorismate mutase, even though these proteins share little homology in the amino acid sequence (9Chook Y.M. Gray J.V. Ke H. Lipscomb W.N. J. Mol. Biol. 1994; 240: 476-500Crossref PubMed Scopus (156) Google Scholar, 10Subramanya H.S. Roper D.I. Dauter D. Dodson E.J. Davies G.J. Wilson K.S. Wigley D.B. Biochemistry. 1996; 35: 792-802Crossref PubMed Scopus (139) Google Scholar, 11Suzuki M. Sugimoto H. Nakagawa A. Tanaka I. Nishihira J. Sakai M. Nat. Struct. Biol. 1996; 3: 259-266Crossref PubMed Scopus (187) Google Scholar, 12Stivers J.T. Abeygunawardana C. Mildvan A.S. Hajipour G. Whitman C.P. Chen L.H. Biochemistry. 1996; 35: 803-813Crossref PubMed Scopus (84) Google Scholar). On the other hand, based on the amino acid sequence homology and structural similarity of MIF withd-dopachrome tautomerase, which convertsd-dopachrome methyl ester to 5,6-dihydroxyindole-2-carboxymethylester, a d-dopachrome tautomerase activity also has been proposed for MIF (13Rosengren E. Bucala R. Arnan P. Jacobsson L. Odh G. Metz C.N. Rorsman H. Mol. Med. 1996; 2: 143-149Crossref PubMed Google Scholar, 14Zhang M. Aman P. Grubb A. Panagopoulos I. Hindemith A. Rosengren E. Rorsman H. FEBS Lett. 1995; 373: 203-206Crossref PubMed Scopus (45) Google Scholar). However, the physiological significance is currently unclear, because natural substrates of MIF have not yet been found. MIF was demonstrated to catalyze the keto-enol isomerization of bothp-hydroxyphenylpyruvate and phenylpyruvate, a hydroxyphenylpyruvate tautomerase activity (15Rosengren E. Aman P. Thelin S. Hansson C. Ahlfors S. Bjork P. Jacobsson L. Rorsman H. FEBS Lett. 1997; 417: 85-88Crossref PubMed Scopus (207) Google Scholar). More recently, MIF has been reported to possess a thiol-protein oxidoreductase activity (16Kleemann R. Kapurniotu A. Frank R.W. Gessner A. Mischke R. Flieger O. Juttner S. Brunner H. Bernhagen J. J. Mol. Biol. 1998; 280: 85-102Crossref PubMed Scopus (267) Google Scholar), which involves the reduction of insulin and 2- hydroxyethyldisulfide. Several lines of evidence suggest that the intracellular redox regulation might be involved in a variety of cellular functions, including cell proliferation, differentiation, tumor promotion, and apoptosis (17Schreck R. Rieber P. Baeuerle P.A. EMBO J. 1991; 10: 2247-2258Crossref PubMed Scopus (3396) Google Scholar, 18Kalebic T. Kinter A. Poli G. Anderson M.E. Meister A. Fauci A.S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 986-990Crossref PubMed Scopus (319) Google Scholar). Antioxidants govern the intracellular redox status. PAG, a known thiol-specific antioxidant, is a member of the peroxiredoxin (Prx) protein family, which was previously referred to as the alkyl hydroperoxide reductase/thiol-specific antioxidant family and is constitutively expressed in most human tissues, but its expression is higher in organs having a higher level of proliferation (19Prosperi M.-T. Ferbus D. Karczinski I. Goubin G. J. Biol. Chem. 1993; 268: 11050-11056Abstract Full Text PDF PubMed Google Scholar, 20Chae H.Z. Robison K. Poole L.B. Church G. Storz G. Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7017-7021Crossref PubMed Scopus (697) Google Scholar). Many Prx proteins are highly conserved in a wide variety of mammalian species such as human, mouse, and bovine, suggesting a biological importance of this type of enzyme (20Chae H.Z. Robison K. Poole L.B. Church G. Storz G. Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7017-7021Crossref PubMed Scopus (697) Google Scholar, 21Chae H.Z. Chung S.J. Rhee S.G. J. Biol. Chem. 1994; 269: 27670-27678Abstract Full Text PDF PubMed Google Scholar, 22Rhee S.G. Chae H.Z. Biofactors. 1994; 4: 177-180PubMed Google Scholar). Most Prx family members contain two conserved cysteines that correspond to Cys47 and Cys170 of yeast thioredoxin peroxidase. In yeast, both Cys47 and Cys170were shown to be necessary for the formation of intermolecular disulfide bonds, and Cys47, but not Cys170, was the primary site of oxidation by H2O2 (23Chae H.Z. Uhm T.B. Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7022-7026Crossref PubMed Scopus (280) Google Scholar). Here we show that PAG binds specifically to MIF in vivo, and we found that this interaction is dependent on the redox status in that the interaction was significantly affected under reducing conditions. Binding of PAG to MIF can repress the d-dopachrome tautomerase activity of MIF. Moreover, this binding resulted in the suppression of the antioxidant activity of PAG. DISCUSSIONThe present study demonstrates that MIF interacts with PAGin vivo, and that the MIF·PAG interaction is dependent on the redox status and the cysteine residues of PAG. In addition, we found that both d-dopachrome tautomerase activity of MIF and antioxidant activity of PAG were negatively regulated by direct association of MIF with PAG.MIF has been proposed as an unusual cytokine containing a combined function both as a conventional cytokine and as an enzyme (1Bernhagen J. Calandra T. Bucala R. J. Mol. Med. 1998; 76: 151-161Crossref PubMed Scopus (155) Google Scholar, 13Rosengren E. Bucala R. Arnan P. Jacobsson L. Odh G. Metz C.N. Rorsman H. Mol. Med. 1996; 2: 143-149Crossref PubMed Google Scholar, 16Kleemann R. Kapurniotu A. Frank R.W. Gessner A. Mischke R. Flieger O. Juttner S. Brunner H. Bernhagen J. J. Mol. Biol. 1998; 280: 85-102Crossref PubMed Scopus (267) Google Scholar). Recently, several studies have reported a variety of enzymatic functions for MIF, including d-dopachrome tautomerase (13Rosengren E. Bucala R. Arnan P. Jacobsson L. Odh G. Metz C.N. Rorsman H. Mol. Med. 1996; 2: 143-149Crossref PubMed Google Scholar, 14Zhang M. Aman P. Grubb A. Panagopoulos I. Hindemith A. Rosengren E. Rorsman H. FEBS Lett. 1995; 373: 203-206Crossref PubMed Scopus (45) Google Scholar), phenylpyruvate tautomerase (15Rosengren E. Aman P. Thelin S. Hansson C. Ahlfors S. Bjork P. Jacobsson L. Rorsman H. FEBS Lett. 1997; 417: 85-88Crossref PubMed Scopus (207) Google Scholar), and a thiol protein oxidoreductase (16Kleemann R. Kapurniotu A. Frank R.W. Gessner A. Mischke R. Flieger O. Juttner S. Brunner H. Bernhagen J. J. Mol. Biol. 1998; 280: 85-102Crossref PubMed Scopus (267) Google Scholar). d-dopachrome tautomerase was discovered during the study of melanin biosynthesis (13Rosengren E. Bucala R. Arnan P. Jacobsson L. Odh G. Metz C.N. Rorsman H. Mol. Med. 1996; 2: 143-149Crossref PubMed Google Scholar). This enzyme was found to have an amino acid sequence that is highly homologous with that of MIF. It has previously been shown that the N-terminal proline of MIF is required for the d-dopachrome tautomerase and phenylpyruvate tautomerase activities (32Swope M. Sun H.W. Blake P.R. Lolis E. EMBO J. 1998; 17: 3534-3541Crossref PubMed Scopus (180) Google Scholar, 33Lubetsky J.B. Swope M. Dealwis C. Blake P. Lolis E. Biochemistry. 1999; 38: 7346-7354Crossref PubMed Scopus (130) Google Scholar, 34Stamps S.L. Fitzgerald M.C. Whitman C.P. Biochemistry. 1998; 37: 10195-10202Crossref PubMed Scopus (71) Google Scholar). However, based on the observed sequence homology with known thiol-protein oxidoreductases (35Puig A. Lyles M.M. Noiva R. Gilbert H.F. J. Biol. Chem. 1994; 269: 19128-19135Abstract Full Text PDF PubMed Google Scholar), it is conceivable that MIF may exhibit a cysteine-dependent enzymatic oxidoreductase activity and that this activity is dependent on the redox-active conserved sequence motif (Cys-X-X-Cys). Analysis of the amino acid sequence of MIF revealed that there was a conserved sequence motif consisting of Cys57-Ala-Leu-Cys60 that was found to be present in the catalytic center of thiol-protein oxidoreductases such as thioredoxin, protein disulfide isomerase, and glutaredoxin. Consistent with this fact, recently a critical role of the conserved cysteine sequence motif (Cys-X-X-Cys) in the oxidoreductase and macrophage-activating activities of MIF was reported (16Kleemann R. Kapurniotu A. Frank R.W. Gessner A. Mischke R. Flieger O. Juttner S. Brunner H. Bernhagen J. J. Mol. Biol. 1998; 280: 85-102Crossref PubMed Scopus (267) Google Scholar). The existence of an intramolecular disulfide bridge was also demonstrated from studies of the conserved cysteine sequence motif, even though previous studies had shown that neither recombinant E. coli-derived MIF nor native MIF from natural cell sources contained an intermolecular disulfide structure (7Bernhagen J. Mitchell R.A. Calandra T. Voelter W. Cerami A. Bucala R. Biochemistry. 1994; 33: 14144-14155Crossref PubMed Scopus (374) Google Scholar, 16Kleemann R. Kapurniotu A. Frank R.W. Gessner A. Mischke R. Flieger O. Juttner S. Brunner H. Bernhagen J. J. Mol. Biol. 1998; 280: 85-102Crossref PubMed Scopus (267) Google Scholar, 36Wistow G.J. Shaughnessy M.P. Lee D.C. Hodin J. Zelenka P.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1272-1275Crossref PubMed Scopus (181) Google Scholar). In addition, MIF was crystallized as a trimer of three identical subunits (11Suzuki M. Sugimoto H. Nakagawa A. Tanaka I. Nishihira J. Sakai M. Nat. Struct. Biol. 1996; 3: 259-266Crossref PubMed Scopus (187) Google Scholar, 28Kato Y. Muto T. Tomura T. Tsumura H. Watarai H. Mikayama T. Ishizaka K. Kuroki R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3007-3010Crossref PubMed Scopus (43) Google Scholar, 29Sun H.W. Bernhagen J. Bucala R. Lolis E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5191-5196Crossref PubMed Scopus (287) Google Scholar). This raises the possibility that MIF may heterodimerize with other cellular proteins in addition to its ability to homodimerize through disulfide linkages between cysteine residues, and that redox regulation may be also involved in the control of this association. To address this question, we sought to identify cellular target proteins that directly associate with MIF. In this study, we report isolation of PAG as an MIF-interacting protein. We performed the coprecipitation experiments of transiently expressed MIF and wild-type PAG, as well as PAG mutants, and demonstrated that MIF associates with wild-type PAG and PAG mutants tested, except for C173S, in mammalian cells (Fig. 2), suggesting that the conserved Cys173 of PAG plays a pivotal role in the formation of intermolecular disulfide linkages between MIF and PAG. However, we cannot rule out the other possibility that the other conserved Cys52 is also involved in the intermolecular disulfide linkages, because we are able to detect a significant decrease in the association of MIF with C52S, although to a somewhat lesser extent, by coprecipitation studies (Fig. 2). To gain more insight into the roles of cysteine residues in the MIF·PAG association, we examined the in vivo binding of MIF and PAG under various redox conditions. As shown in Fig. 3, upon reducing conditions the association was remarkably decreased in a dose-dependent manner, suggesting that the in vivo association of MIF and PAG may be mediated through disulfide linkages of cysteine residues. Recent studies have suggested that MIF and thiol antioxidants could be bridged through inter-chain disulfides (24Jin D.-Y. Chae H.Z. Rhee S.G. Jeang K.-T. J. Biol. Chem. 1997; 272: 30952-30961Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar, 37Jaschke A. Mi H. Tropschug M. J. Mol. Biol. 1998; 277: 763-769Crossref PubMed Scopus (57) Google Scholar, 38Swope M.D. Sun H.-W. Klockow B. Blake P. Lolis E. J. Biol. Chem. 1998; 273: 14877-14884Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). It is necessary to determine whether the conserved cysteine residues of PAG could influence homo- and heterodimerization and whether this complex formation contributes toward the regulation of functional activities of interacting partners.To understand how the interaction contributes to the activity of the respective proteins, the examination of how MIF or PAG binding modulates the interacting protein function seems to be important. As shown in this report, it can be concluded that PAG is a negative regulator of MIF activity. Moreover, we confirmed this observation with the PAG mutants such as C52S, C71S, and C173S. From these mutation experiments, all mutants, except for C173S, resulted in a decrease ofd-dopachrome tautomerase activity of MIF (Fig. 4 and data not shown). These findings can be explained either by a physical binding of PAG to MIF or by an enzymatic function of PAG. However, the fact that C52S mutant, but not C173S, indeed decreases the MIF activity, together with our observation that both Cys52 and Cys173 are equally critical for the antioxidant activity of PAG (data not shown), does not favor the second model describing the importance of the enzymatic function of PAG in the regulation of MIF activity. In addition, it has recently been shown that several MIF residues near the N-terminal proline are perturbed upon addition ofS-hexylglutathione and affected byp-hydroxyphenylpyruvate, a substrate for the phenylpyruvate tautomerase activity of MIF (33Lubetsky J.B. Swope M. Dealwis C. Blake P. Lolis E. Biochemistry. 1999; 38: 7346-7354Crossref PubMed Scopus (130) Google Scholar, 38Swope M.D. Sun H.-W. Klockow B. Blake P. Lolis E. J. Biol. Chem. 1998; 273: 14877-14884Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Because our studies reported here demonstrated that PAG could inhibit the d-dopachrome tautomerase activity of MIF, it is tempting to speculate that this effect is mediated through the perturbation of the residues surrounding the N-terminal proline on the three-dimensional structure of MIF by direct binding of PAG. Based on our observed results, we imagine that rather than direct contact with the residues surrounding the N-terminal proline, the conformational effect that is caused by intermolecular disulfide linkages between cysteine residues of PAG and MIF likely plays a role that is important in the regulation of MIFd-dopachrome tautomerase activity, probably by structural changes within the region near the N-terminal proline.A recent study (30Kleemann R. Mischke R. Kapurniotu A. Brunner H. Bernhagen J. FEBS Lett. 1998; 430: 191-196Crossref PubMed Scopus (61) Google Scholar) demonstrated that Aop1, a human thioredoxin peroxidase, and cyclophilin18 do not only interact physically, but binding of cyclophilin18 also stimulates the antioxidant activity of Aop1, suggesting an important role for the direct interaction in the regulation of the antioxidant activity of thioredoxin peroxidases. To test a possible influence of binding of MIF on the enzymatic activity of PAG, we used a similar approach. As shown in Fig. 5, a dramatic decrease was observed in the antioxidant activity of PAG when increasing amounts of MIF were added to the PAG. This also indicates that MIF negatively regulates the PAG activity. In this regard, the mechanism of interaction of MIF and PAG will be the interest of future study. Further understanding of the mechanism of this interaction will result from the identification of other interacting proteins associated with MIF or PAG and detailed analyses of the binding region in the MIF·PAG interaction. The present study demonstrates that MIF interacts with PAGin vivo, and that the MIF·PAG interaction is dependent on the redox status and the cysteine residues of PAG. In addition, we found that both d-dopachrome tautomerase activity of MIF and antioxidant activity of PAG were negatively regulated by direct association of MIF with PAG. MIF has been proposed as an unusual cytokine containing a combined function both as a conventional cytokine and as an enzyme (1Bernhagen J. Calandra T. Bucala R. J. Mol. Med. 1998; 76: 151-161Crossref PubMed Scopus (155) Google Scholar, 13Rosengren E. Bucala R. Arnan P. Jacobsson L. Odh G. Metz C.N. Rorsman H. Mol. Med. 1996; 2: 143-149Crossref PubMed Google Scholar, 16Kleemann R. Kapurniotu A. Frank R.W. Gessner A. Mischke R. Flieger O. Juttner S. Brunner H. Bernhagen J. J. Mol. Biol. 1998; 280: 85-102Crossref PubMed Scopus (267) Google Scholar). Recently, several studies have reported a variety of enzymatic functions for MIF, including d-dopachrome tautomerase (13Rosengren E. Bucala R. Arnan P. Jacobsson L. Odh G. Metz C.N. Rorsman H. Mol. Med. 1996; 2: 143-149Crossref PubMed Google Scholar, 14Zhang M. Aman P. Grubb A. Panagopoulos I. Hindemith A. Rosengren E. Rorsman H. FEBS Lett. 1995; 373: 203-206Crossref PubMed Scopus (45) Google Scholar), phenylpyruvate tautomerase (15Rosengren E. Aman P. Thelin S. Hansson C. Ahlfors S. Bjork P. Jacobsson L. Rorsman H. FEBS Lett. 1997; 417: 85-88Crossref PubMed Scopus (207) Google Scholar), and a thiol protein oxidoreductase (16Kleemann R. Kapurniotu A. Frank R.W. Gessner A. Mischke R. Flieger O. Juttner S. Brunner H. Bernhagen J. J. Mol. Biol. 1998; 280: 85-102Crossref PubMed Scopus (267) Google Scholar). d-dopachrome tautomerase was discovered during the study of melanin biosynthesis (13Rosengren E. Bucala R. Arnan P. Jacobsson L. Odh G. Metz C.N. Rorsman H. Mol. Med. 1996; 2: 143-149Crossref PubMed Google Scholar). This enzyme was found to have an amino acid sequence that is highly homologous with that of MIF. It has previously been shown that the N-terminal proline of MIF is required for the d-dopachrome tautomerase and phenylpyruvate tautomerase activities (32Swope M. Sun H.W. Blake P.R. Lolis E. EMBO J. 1998; 17: 3534-3541Crossref PubMed Scopus (180) Google Scholar, 33Lubetsky J.B. Swope M. Dealwis C. Blake P. Lolis E. Biochemistry. 1999; 38: 7346-7354Crossref PubMed Scopus (130) Google Scholar, 34Stamps S.L. Fitzgerald M.C. Whitman C.P. Biochemistry. 1998; 37: 10195-10202Crossref PubMed Scopus (71) Google Scholar). However, based on the observed sequence homology with known thiol-protein oxidoreductases (35Puig A. Lyles M.M. Noiva R. Gilbert H.F. J. Biol. Chem. 1994; 269: 19128-19135Abstract Full Text PDF PubMed Google Scholar), it is conceivable that MIF may exhibit a cysteine-dependent enzymatic oxidoreductase activity and that this activity is dependent on the redox-active conserved sequence motif (Cys-X-X-Cys). Analysis of the amino acid sequence of MIF revealed that there was a conserved sequence motif consisting of Cys57-Ala-Leu-Cys60 that was found to be present in the catalytic center of thiol-protein oxidoreductases such as thioredoxin, protein disulfide isomerase, and glutaredoxin. Consistent with this fact, recently a critical role of the conserved cysteine sequence motif (Cys-X-X-Cys) in the oxidoreductase and macrophage-activating activities of MIF was reported (16Kleemann R. Kapurniotu A. Frank R.W. Gessner A. Mischke R. Flieger O. Juttner S. Brunner H. Bernhagen J. J. Mol. Biol. 1998; 280: 85-102Crossref PubMed Scopus (267) Google Scholar). The existence of an intramolecular disulfide bridge was also demonstrated from studies of the conserved cysteine sequence motif, even though previous studies had shown that neither recombinant E. coli-derived MIF nor native MIF from natural cell sources contained an intermolecular disulfide structure (7Bernhagen J. Mitchell R.A. Calandra T. Voelter W. Cerami A. Bucala R. Biochemistry. 1994; 33: 14144-14155Crossref PubMed Scopus (374) Google Scholar, 16Kleemann R. Kapurniotu A. Frank R.W. Gessner A. Mischke R. Flieger O. Juttner S. Brunner H. Bernhagen J. J. Mol. Biol. 1998; 280: 85-102Crossref PubMed Scopus (267) Google Scholar, 36Wistow G.J. Shaughnessy M.P. Lee D.C. Hodin J. Zelenka P.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1272-1275Crossref PubMed Scopus (181) Google Scholar). In addition, MIF was crystallized as a trimer of three identical subunits (11Suzuki M. Sugimoto H. Nakagawa A. Tanaka I. Nishihira J. Sakai M. Nat. Struct. Biol. 1996; 3: 259-266Crossref PubMed Scopus (187) Google Scholar, 28Kato Y. Muto T. Tomura T. Tsumura H. Watarai H. Mikayama T. Ishizaka K. Kuroki R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3007-3010Crossref PubMed Scopus (43) Google Scholar, 29Sun H.W. Bernhagen J. Bucala R. Lolis E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5191-5196Crossref PubMed Scopus (287) Google Scholar). This raises the possibility that MIF may heterodimerize with other cellular proteins in addition to its ability to homodimerize through disulfide linkages between cysteine residues, and that redox regulation may be also involved in the control of this association. To address this question, we sought to identify cellular target proteins that directly associate with MIF. In this study, we report isolation of PAG as an MIF-interacting protein. We performed the coprecipitation experiments of transiently expressed MIF and wild-type PAG, as well as PAG mutants, and demonstrated that MIF associates with wild-type PAG and PAG mutants tested, except for C173S, in mammalian cells (Fig. 2), suggesting that the conserved Cys173 of PAG plays a pivotal role in the formation of intermolecular disulfide linkages between MIF and PAG. However, we cannot rule out the other possibility that the other conserved Cys52 is also involved in the intermolecular disulfide linkages, because we are able to detect a significant decrease in the association of MIF with C52S, although to a somewhat lesser extent, by coprecipitation studies (Fig. 2). To gain more insight into the roles of cysteine residues in the MIF·PAG association, we examined the in vivo binding of MIF and PAG under various redox conditions. As shown in Fig. 3, upon reducing conditions the association was remarkably decreased in a dose-dependent manner, suggesting that the in vivo association of MIF and PAG may be mediated through disulfide linkages of cysteine residues. Recent studies have suggested that MIF and thiol antioxidants could be bridged through inter-chain disulfides (24Jin D.-Y. Chae H.Z. Rhee S.G. Jeang K.-T. J. Biol. Chem. 1997; 272: 30952-30961Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar, 37Jaschke A. Mi H. Tropschug M. J. Mol. Biol. 1998; 277: 763-769Crossref PubMed Scopus (57) Google Scholar, 38Swope M.D. Sun H.-W. Klockow B. Blake P. Lolis E. J. Biol. Chem. 1998; 273: 14877-14884Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). It is necessary to determine whether the conserved cysteine residues of PAG could influence homo- and heterodimerization and whether this complex formation contributes toward the regulation of functional activities of interacting partners. To understand how the interaction contributes to the activity of the respective proteins, the examination of how MIF or PAG binding modulates the interacting protein function seems to be important. As shown in this report, it can be concluded that PAG is a negative regulator of MIF activity. Moreover, we confirmed this observation with the PAG mutants such as C52S, C71S, and C173S. From these mutation experiments, all mutants, except for C173S, resulted in a decrease ofd-dopachrome tautomerase activity of MIF (Fig. 4 and data not shown). These findings can be explained either by a physical binding of PAG to MIF or by an enzymatic function of PAG. However, the fact that C52S mutant, but not C173S, indeed decreases the MIF activity, together with our observation that both Cys52 and Cys173 are equally critical for the antioxidant activity of PAG (data not shown), does not favor the second model describing the importance of the enzymatic function of PAG in the regulation of MIF activity. In addition, it has recently been shown that several MIF residues near the N-terminal proline are perturbed upon addition ofS-hexylglutathione and affected byp-hydroxyphenylpyruvate, a substrate for the phenylpyruvate tautomerase activity of MIF (33Lubetsky J.B. Swope M. Dealwis C. Blake P. Lolis E. Biochemistry. 1999; 38: 7346-7354Crossref PubMed Scopus (130) Google Scholar, 38Swope M.D. Sun H.-W. Klockow B. Blake P. Lolis E. J. Biol. Chem. 1998; 273: 14877-14884Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Because our studies reported here demonstrated that PAG could inhibit the d-dopachrome tautomerase activity of MIF, it is tempting to speculate that this effect is mediated through the perturbation of the residues surrounding the N-terminal proline on the three-dimensional structure of MIF by direct binding of PAG. Based on our observed results, we imagine that rather than direct contact with the residues surrounding the N-terminal proline, the conformational effect that is caused by intermolecular disulfide linkages between cysteine residues of PAG and MIF likely plays a role that is important in the regulation of MIFd-dopachrome tautomerase activity, probably by structural changes within the region near the N-terminal proline. A recent study (30Kleemann R. Mischke R. Kapurniotu A. Brunner H. Bernhagen J. FEBS Lett. 1998; 430: 191-196Crossref PubMed Scopus (61) Google Scholar) demonstrated that Aop1, a human thioredoxin peroxidase, and cyclophilin18 do not only interact physically, but binding of cyclophilin18 also stimulates the antioxidant activity of Aop1, suggesting an important role for the direct interaction in the regulation of the antioxidant activity of thioredoxin peroxidases. To test a possible influence of binding of MIF on the enzymatic activity of PAG, we used a similar approach. As shown in Fig. 5, a dramatic decrease was observed in the antioxidant activity of PAG when increasing amounts of MIF were added to the PAG. This also indicates that MIF negatively regulates the PAG activity. In this regard, the mechanism of interaction of MIF and PAG will be the interest of future study. Further understanding of the mechanism of this interaction will result from the identification of other interacting proteins associated with MIF or PAG and detailed analyses of the binding region in the MIF·PAG interaction. We thank Dr. I. H. Kim, Department of Biochemistry, Paichai University, for helpful instructions on GS protection assay." @default.
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W1991821808 title "Regulation of Macrophage Migration Inhibitory Factor and Thiol-specific Antioxidant Protein PAG by Direct Interaction" @default.
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