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

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 Cys 173 of PAG is responsible for the formation of MIF⅐PAG complex. In addition, the interaction was highly dependent on the reducing conditions such as dithiothreitol or ␤-mercaptoethanol but not in the presence of H 2 O 2 . 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 of D-dopachrome tautomerase activity of MIF. Moreover, MIF has been shown to bind to PAG and it also inhibits the antioxidant activity of PAG.

Allosteric inhibition of macrophage migration inhibitory factor revealed by ibudilast

“…For example, the substrate HPP used to measure the K i is a pseudosubstrate because the K m is more than 1,000-fold higher than the in vivo concentration of HPP (20,29). MIF has been reported to form complexes with over a dozen proteins (15,(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40). Potential interactions between the enzyme and physiological molecules (substrate or protein) may enhance the affinity of the allosteric inhibitors for MIF and result in their robust chemotactic inhibitory effects.…”

Human Umbilical Cord Blood Cells Protect Oligodendrocytes from Brain Ischemia through Akt Signal Transduction

“…Their actions have not only been implicated in neurodegenerative diseases but also as a target for cancer treatment and immune functioning (37)(38)(39)(40). Administration of peroxiredoxin 3 protected neurons from ischemic insult resulting in enhanced behavioral recovery (41).…”

Regulation of Transforming Growth Factor-β Signaling and PDK1 Kinase Activity by Physical Interaction between PDK1 and Serine-Threonine Kinase Receptor-associated Protein

“…Cell Culture, Plasmids, and Reagents-293T, HepG2, Hep3B, and SK-N-BE(2)C cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS (Invitrogen) as described (26). The eukaryotic glutathione S-transferase (GST) expression vector (pEBG) and pFLAG-CMV-2 vector with a FLAG epitope were obtained as described previously (27).…”

“…The reverse primers for FLAG-PDK1(PH) (5Ј-GCCTCGAGTCACTGCACAGCGGCGTC-3Ј) and FLAG-PDK-1(CA) (5Ј-GCCAGCTGGGTGAGCTTCGGAGGCGT-3Ј) contain an XhoI and a SalI site (underlined). The amplified PCR products for deletion mutants were cut with EcoRI plus XhoI and EcoRI plus SalI, and cloned into pFLAG-PAG (26), a PAG cDNA cloned into pFLAG-C-MV-2 vector, to generate the FLAG-PDK1(PH) and FLAG-PDK1(CA) constructs, respectively. The identity of all PCR products was confirmed by nucleotide sequencing analysis on both strands.…”