Regulation of Macrophage Migration Inhibitory Factor and Thiol-specific Antioxidant Protein PAG by Direct Interaction

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 activity R. A. A. R. S. H. Bernhagen J. J. Mol. Biol. 1998; PubMed Scopus Google which the of and of that the regulation be involved in a of and R. P. J. PubMed Scopus Google Scholar, T. A. G. A. A.S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). the PAG, a thiol-specific is a of the peroxiredoxin which was to as the antioxidant and is in but is in a of D. I. G. J. Biol. PubMed Google Scholar, K. G. G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar). proteins highly in a of such as and suggesting a of this of K. G. G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar, J. Biol. 1994; PubMed Google Scholar, 1994; Google Scholar). that to and of yeast In and shown to be for the formation of disulfide and but not was the of by Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google we that PAG to MIF in and we found that this interaction is dependent on the in that the interaction was significantly affected reducing of PAG to MIF the d-dopachrome tautomerase activity of MIF. Moreover, this in the of the antioxidant activity of that MIF with and that the MIF·PAG interaction is dependent on the and the of PAG. In addition, we found that d-dopachrome tautomerase activity of MIF and antioxidant activity of PAG by direct association of MIF with has been proposed as an cytokine a as a cytokine and as an (1Bernhagen J. Calandra T. Bucala R. J. Mol. Med. 1998; 76: 151-161Crossref PubMed Scopus (155) Google Scholar, E. Bucala R. Arnan P. Jacobsson L. Odh G. Metz C.N. Rorsman H. Mol. Med. 1996; 2: 143-149Crossref PubMed Google Scholar, R. A. A. R. S. H. Bernhagen J. J. Mol. Biol. 1998; PubMed Scopus Google Scholar). studies have reported a of for 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 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 and a R. A. A. R. S. H. Bernhagen J. J. Mol. Biol. 1998; PubMed Scopus Google Scholar). d-dopachrome tautomerase was the of (13Rosengren E. Bucala R. Arnan P. Jacobsson L. Odh G. Metz C.N. Rorsman H. Mol. Med. 1996; 2: 143-149Crossref PubMed Google Scholar). was found to have an amino acid sequence that is highly with that of MIF. has been shown that the of MIF is for the d-dopachrome tautomerase and tautomerase activities M. E. J. 1998; 17: PubMed Scopus Google Scholar, M. C. P. E. Biochemistry. PubMed Scopus Google Scholar, Whitman C.P. Biochemistry. 1998; PubMed Scopus Google Scholar). However, based on the sequence homology with thiol-protein A. R. J. Biol. 1994; PubMed Google it is that MIF may a activity and that this activity is dependent on the sequence Analysis of the amino acid sequence of MIF revealed that was a sequence of that was found to be in the of thiol-protein such as disulfide and with this a role of the sequence in the and activities of MIF was reported R. A. A. R. S. H. Bernhagen J. J. Mol. Biol. 1998; PubMed Scopus Google Scholar). The of an disulfide was also demonstrated from studies of the sequence even though studies shown that E. MIF MIF from natural an disulfide (7Bernhagen J. Mitchell R.A. Calandra T. Voelter W. Cerami A. Bucala R. Biochemistry. 1994; 33: 14144-14155Crossref PubMed Scopus (374) Google Scholar, R. A. A. R. S. H. Bernhagen J. J. Mol. Biol. 1998; PubMed Scopus Google Scholar, G.J. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). In addition, MIF was as a of M. Sugimoto H. Nakagawa A. Tanaka I. Nishihira J. Sakai M. Nat. Struct. Biol. 1996; 3: 259-266Crossref PubMed Scopus (187) Google Scholar, T. T. H. H. T. Ishizaka K. R. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, Bernhagen J. Bucala R. E. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). the that MIF may with other proteins in to to disulfide and that regulation may be also involved in the control of this To this we to proteins that with MIF. In this we of PAG as an the of transiently MIF and wild-type PAG, as as PAG and demonstrated that MIF with wild-type PAG and PAG mutants for C173S, in cells suggesting that the Cys173 of PAG plays a role in the formation of disulfide MIF and PAG. However, we the other that the other is also involved in the disulfide because we to a in the association of MIF with C52S, to a by studies To the of in the MIF·PAG we the in of MIF and PAG shown in reducing conditions the association was decreased in a dose-dependent suggesting that the in association of MIF and PAG may be disulfide of studies have that MIF and be J. Biol. 1997; PubMed Scopus Google Scholar, A. H. M. J. Mol. Biol. 1998; PubMed Scopus Google Scholar, P. E. J. Biol. 1998; PubMed Scopus Google Scholar). is to the of PAG and and this formation the regulation of activities of interacting the interaction to the activity of the the of MIF or PAG the interacting to be shown in this it be that PAG is a of MIF Moreover, we this with the PAG mutants such as C52S, C71S, and these for C173S, in a ofd-dopachrome tautomerase activity of MIF and not be by a of PAG to MIF or by an of PAG. However, the that C52S but not C173S, the MIF with that and Cys173 for the antioxidant activity of PAG not not the the of the of PAG in the regulation of MIF In addition, it has been shown that MIF the and affected a for the tautomerase activity of MIF M. C. P. E. Biochemistry. PubMed Scopus Google Scholar, P. E. J. Biol. 1998; PubMed Scopus Google Scholar). studies reported demonstrated that PAG the d-dopachrome tautomerase activity of it is to that this is the of the the on the of MIF by direct of PAG. on we that direct with the the the that is by disulfide of PAG and MIF plays a role that is important in the regulation of tautomerase by structural the the R. R. A. H. Bernhagen J. FEBS Lett. 1998; PubMed Scopus Google demonstrated that a and not but of also the antioxidant activity of suggesting an important role for the direct interaction in the regulation of the antioxidant activity of To a of of MIF on the activity of PAG, we used a shown in a was in the antioxidant activity of PAG of MIF to the PAG. also that MIF the PAG In this the mechanism of interaction of MIF and PAG be the of of the mechanism of this interaction from the of other interacting proteins with MIF or PAG and of the in the MIF·PAG 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 activity R. A. A. R. S. H. Bernhagen J. J. Mol. Biol. 1998; PubMed Scopus Google which the of and of that the regulation be involved in a of and R. P. J. PubMed Scopus Google Scholar, T. A. G. A. A.S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). the PAG, a thiol-specific is a of the peroxiredoxin which was to as the antioxidant and is in but is in a of D. I. G. J. Biol. PubMed Google Scholar, K. G. G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar). proteins highly in a of such as and suggesting a of this of K. G. G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar, J. Biol. 1994; PubMed Google Scholar, 1994; Google Scholar). that to and of yeast In and shown to be for the formation of disulfide and but not was the of by Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar). we that PAG to MIF in and we found that this interaction is dependent on the in that the interaction was significantly affected reducing of PAG to MIF the d-dopachrome tautomerase activity of MIF. Moreover, this in the of the antioxidant activity of PAG. that MIF with and that the MIF·PAG interaction is dependent on the and the of PAG. In addition, we found that d-dopachrome tautomerase activity of MIF and antioxidant activity of PAG by direct association of MIF with has been proposed as an cytokine a as a cytokine and as an (1Bernhagen J. Calandra T. Bucala R. J. Mol. Med. 1998; 76: 151-161Crossref PubMed Scopus (155) Google Scholar, E. Bucala R. Arnan P. Jacobsson L. Odh G. Metz C.N. Rorsman H. Mol. Med. 1996; 2: 143-149Crossref PubMed Google Scholar, R. A. A. R. S. H. Bernhagen J. J. Mol. Biol. 1998; PubMed Scopus Google Scholar). studies have reported a of for 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 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 and a R. A. A. R. S. H. Bernhagen J. J. Mol. Biol. 1998; PubMed Scopus Google Scholar). d-dopachrome tautomerase was the of (13Rosengren E. Bucala R. Arnan P. Jacobsson L. Odh G. Metz C.N. Rorsman H. Mol. Med. 1996; 2: 143-149Crossref PubMed Google Scholar). was found to have an amino acid sequence that is highly with that of MIF. has been shown that the of MIF is for the d-dopachrome tautomerase and tautomerase activities M. E. J. 1998; 17: PubMed Scopus Google Scholar, M. C. P. E. Biochemistry. PubMed Scopus Google Scholar, Whitman C.P. Biochemistry. 1998; PubMed Scopus Google Scholar). However, based on the sequence homology with thiol-protein A. R. J. Biol. 1994; PubMed Google it is that MIF may a activity and that this activity is dependent on the sequence Analysis of the amino acid sequence of MIF revealed that was a sequence of that was found to be in the of thiol-protein such as disulfide and with this a role of the sequence in the and activities of MIF was reported R. A. A. R. S. H. Bernhagen J. J. Mol. Biol. 1998; PubMed Scopus Google Scholar). The of an disulfide was also demonstrated from studies of the sequence even though studies shown that E. MIF MIF from natural an disulfide (7Bernhagen J. Mitchell R.A. Calandra T. Voelter W. Cerami A. Bucala R. Biochemistry. 1994; 33: 14144-14155Crossref PubMed Scopus (374) Google Scholar, R. A. A. R. S. H. Bernhagen J. J. Mol. Biol. 1998; PubMed Scopus Google Scholar, G.J. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). In addition, MIF was as a of M. Sugimoto H. Nakagawa A. Tanaka I. Nishihira J. Sakai M. Nat. Struct. Biol. 1996; 3: 259-266Crossref PubMed Scopus (187) Google Scholar, T. T. H. H. T. Ishizaka K. R. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, Bernhagen J. Bucala R. E. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). the that MIF may with other proteins in to to disulfide and that regulation may be also involved in the control of this To this we to proteins that with MIF. In this we of PAG as an the of transiently MIF and wild-type PAG, as as PAG and demonstrated that MIF with wild-type PAG and PAG mutants for C173S, in cells suggesting that the Cys173 of PAG plays a role in the formation of disulfide MIF and PAG. However, we the other that the other is also involved in the disulfide because we to a in the association of MIF with C52S, to a by studies To the of in the MIF·PAG we the in of MIF and PAG shown in reducing conditions the association was decreased in a dose-dependent suggesting that the in association of MIF and PAG may be disulfide of studies have that MIF and be J. Biol. 1997; PubMed Scopus Google Scholar, A. H. M. J. Mol. Biol. 1998; PubMed Scopus Google Scholar, P. E. J. Biol. 1998; PubMed Scopus Google Scholar). is to the of PAG and and this formation the regulation of activities of interacting the interaction to the activity of the the of MIF or PAG the interacting to be shown in this it be that PAG is a of MIF Moreover, we this with the PAG mutants such as C52S, C71S, and these for C173S, in a ofd-dopachrome tautomerase activity of MIF and not be by a of PAG to MIF or by an of PAG. However, the that C52S but not C173S, the MIF with that and Cys173 for the antioxidant activity of PAG not not the the of the of PAG in the regulation of MIF In addition, it has been shown that MIF the and affected a for the tautomerase activity of MIF M. C. P. E. Biochemistry. PubMed Scopus Google Scholar, P. E. J. Biol. 1998; PubMed Scopus Google Scholar). studies reported demonstrated that PAG the d-dopachrome tautomerase activity of it is to that this is the of the the on the of MIF by direct of PAG. on we that direct with the the the that is by disulfide of PAG and MIF plays a role that is important in the regulation of tautomerase by structural the the R. R. A. H. Bernhagen J. FEBS Lett. 1998; PubMed Scopus Google demonstrated that a and not but of also the antioxidant activity of suggesting an important role for the direct interaction in the regulation of the antioxidant activity of To a of of MIF on the activity of PAG, we used a shown in a was in the antioxidant activity of PAG of MIF to the PAG. also that MIF the PAG In this the mechanism of interaction of MIF and PAG be the of of the mechanism of this interaction from the of other interacting proteins with MIF or PAG and of the in the MIF·PAG The that MIF with and that the MIF·PAG interaction is dependent on the and the of PAG. In addition, we found that d-dopachrome tautomerase activity of MIF and antioxidant activity of PAG by direct association of MIF with PAG. MIF has been proposed as an cytokine a as a cytokine and as an (1Bernhagen J. Calandra T. Bucala R. J. Mol. Med. 1998; 76: 151-161Crossref PubMed Scopus (155) Google Scholar, E. Bucala R. Arnan P. Jacobsson L. Odh G. Metz C.N. Rorsman H. Mol. Med. 1996; 2: 143-149Crossref PubMed Google Scholar, R. A. A. R. S. H. Bernhagen J. J. Mol. Biol. 1998; PubMed Scopus Google Scholar). studies have reported a of for 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 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 and a R. A. A. R. S. H. Bernhagen J. J. Mol. Biol. 1998; PubMed Scopus Google Scholar). d-dopachrome tautomerase was the of (13Rosengren E. Bucala R. Arnan P. Jacobsson L. Odh G. Metz C.N. Rorsman H. Mol. Med. 1996; 2: 143-149Crossref PubMed Google Scholar). was found to have an amino acid sequence that is highly with that of MIF. has been shown that the of MIF is for the d-dopachrome tautomerase and tautomerase activities M. E. J. 1998; 17: PubMed Scopus Google Scholar, M. C. P. E. Biochemistry. PubMed Scopus Google Scholar, Whitman C.P. Biochemistry. 1998; PubMed Scopus Google Scholar). However, based on the sequence homology with thiol-protein A. R. J. Biol. 1994; PubMed Google it is that MIF may a activity and that this activity is dependent on the sequence Analysis of the amino acid sequence of MIF revealed that was a sequence of that was found to be in the of thiol-protein such as disulfide and with this a role of the sequence in the and activities of MIF was reported R. A. A. R. S. H. Bernhagen J. J. Mol. Biol. 1998; PubMed Scopus Google Scholar). The of an disulfide was also demonstrated from studies of the sequence even though studies shown that E. MIF MIF from natural an disulfide (7Bernhagen J. Mitchell R.A. Calandra T. Voelter W. Cerami A. Bucala R. Biochemistry. 1994; 33: 14144-14155Crossref PubMed Scopus (374) Google Scholar, R. A. A. R. S. H. Bernhagen J. J. Mol. Biol. 1998; PubMed Scopus Google Scholar, G.J. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). In addition, MIF was as a of M. Sugimoto H. Nakagawa A. Tanaka I. Nishihira J. Sakai M. Nat. Struct. Biol. 1996; 3: 259-266Crossref PubMed Scopus (187) Google Scholar, T. T. H. H. T. Ishizaka K. R. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, Bernhagen J. Bucala R. E. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). the that MIF may with other proteins in to to disulfide and that regulation may be also involved in the control of this To this we to proteins that with MIF. In this we of PAG as an the of transiently MIF and wild-type PAG, as as PAG and demonstrated that MIF with wild-type PAG and PAG mutants for C173S, in cells suggesting that the Cys173 of PAG plays a role in the formation of disulfide MIF and PAG. However, we the other that the other is also involved in the disulfide because we to a in the association of MIF with C52S, to a by studies To the of in the MIF·PAG we the in of MIF and PAG shown in reducing conditions the association was decreased in a dose-dependent suggesting that the in association of MIF and PAG may be disulfide of studies have that MIF and be J. Biol. 1997; PubMed Scopus Google Scholar, A. H. M. J. Mol. Biol. 1998; PubMed Scopus Google Scholar, P. E. J. Biol. 1998; PubMed Scopus Google Scholar). is to the of PAG and and this formation the regulation of activities of interacting To the interaction to the activity of the the of MIF or PAG the interacting to be shown in this it be that PAG is a of MIF Moreover, we this with the PAG mutants such as C52S, C71S, and these for C173S, in a ofd-dopachrome tautomerase activity of MIF and not be by a of PAG to MIF or by an of PAG. However, the that C52S but not C173S, the MIF with that and Cys173 for the antioxidant activity of PAG not not the the of the of PAG in the regulation of MIF In addition, it has been shown that MIF the and affected a for the tautomerase activity of MIF M. C. P. E. Biochemistry. PubMed Scopus Google Scholar, P. E. J. Biol. 1998; PubMed Scopus Google Scholar). studies reported demonstrated that PAG the d-dopachrome tautomerase activity of it is to that this is the of the the on the of MIF by direct of PAG. on we that direct with the the the that is by disulfide of PAG and MIF plays a role that is important in the regulation of tautomerase by structural the the R. R. A. H. Bernhagen J. FEBS Lett. 1998; PubMed Scopus Google demonstrated that a and not but of also the antioxidant activity of suggesting an important role for the direct interaction in the regulation of the antioxidant activity of To a of of MIF on the activity of PAG, we used a shown in a was in the antioxidant activity of PAG of MIF to the PAG. also that MIF the PAG In this the mechanism of interaction of MIF and PAG be the of of the mechanism of this interaction from the of other interacting proteins with MIF or PAG and of the in the MIF·PAG I. H. of for on