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 res ponse. To investigate the molecular mechanism of MIF action, we have used t he 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 mut ants (C52S, C71S, and C173S) revealed that this association was significant ly affected by C173S, but not C52S and C71S, indicating that a disulfide in volving Cys(173) Of PAG is responsible for the formation of MIF.PAG complex . In addition, the interaction was highly dependent on the reducing conditi ons such as dithiothreitol or beta -mercaptoethanol but not in the presence of H2O2. Analysis of the activities of the interacting proteins showed tha t the D-dopachrome tautomerase activity of MIF was decreased in a dose-depe ndent manner by coexpression of wild-type PAG, C52S, and C71S, whereas C173 S was almost ineffective, suggesting that the direct interaction may be inv olved in the control of D-dopachrome tautomerase activity of MIF. Moreover, MIF has been shown to bind to PAG and it also inhibits the antioxidant act ivity of PAG.