Specific reduction of insulin disulfides by macrophage migration inhibitory factor (MIF) with glutathione and dihydrolipoamide: potential role in cellular redox processes.
The molecular mechanism of action of MIF, a cytokine that plays a critical role in the host immune and inflammatory response, has not yet been identified. We recently demonstrated that MIF is an enzyme that exhibits oxidoreductase activity by a cysteine thiol-mediated mechanism. Here we further investigated this function by examining the reduction of insulin disulfides by wild-type human MIF (wtMIF) using various substrates, namely glutathione (GSH), dihydrolipoamide, L-cysteine, beta-mercaptoethanol and dithiothreitol. The activity of wtMIF was compared to that of the relevant cysteine mutants of MIF and to two carboxy-truncated mutants. Only GSH and dihydrolipoamide were found to serve as reductants, whereas the other substrates were not utilized by MIF. Reduction of insulin disulfides by MIF was closely dependent on the presence of the Cys57-Ala-Leu-Cys60 (CALC) motif-forming cysteines C57 and C60, whereas C81 was not involved (activities: 51+/-13%, 14+/-5%, and 70+/-12% of wtMIF, respectively, and 20+/-3% for the double mutant C57S/C60S). Confirming the notion that the activity of MIF was dependent on the CALC motif in the central region of the MIF sequence, the C-terminal deletion mutants MIF(1-105) and MIF(1-110) were found to be fully active. The favored use of GSH and dihydrolipoamide indicated that MIF may be involved in the regulation of cellular redox processes and was supported further by the finding that MIF expression by the cell lines COS-1 and RAW 264.7 was significantly induced upon treatment with the oxidant hydrogen peroxide.
Kleemann
R
R
Laboratory of Biochemistry, University of Stuttgart, Fraunhofer Institute/Fh-IGB, Germany.
Mischke
R
R
Kapurniotu
A
A
Brunner
H
H
Bernhagen
J
J
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D005978:Glutathione / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007328:Insulin / Q000737:chemistry*; D008263:Macrophage Migration-Inhibitory Factors / Q000737:chemistry* / Q000235:genetics; D008264:Macrophages; D008969:Molecular Sequence Data; D009154:Mutation; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D019163:Reducing Agents; D017384:Sequence Deletion; D013379:Substrate Specificity; D008063:Thioctic Acid / Q000031:analogs & derivatives / Q000737:chemistry
D004220:Disulfides; D007328:Insulin; D008263:Macrophage Migration-Inhibitory Factors; D016877:Oxidants; D019163:Reducing Agents; C007409:dihydrolipoamide; D008063:Thioctic Acid; D006861:Hydrogen Peroxide; D010088:Oxidoreductases; D005978:Glutathione; D003545:Cysteine