Role of the Cysteine 81 Residue of Macrophage Migration Inhibitory Factor as a Molecular Redox Switch

Abstract

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory and tumor-promoting cytokine that occurs in two redox-dependent immunologically distinct conformational isoforms. The disease-related structural isoform of MIF (oxMIF) can be specifically and predominantly detected in the circulation of patients with inflammatory diseases and in tumor tissue, whereas the ubiquitously expressed isoform of MIF (redMIF) is abundantly expressed in healthy and diseased subjects. In this article, we report that cysteine 81 within MIF serves as a "switch cysteine" for the conversion of redMIF to oxMIF. Modulating cysteine 81 by thiol reactive agents leads to significant structural rearrangements of the protein, resulting in a decreased β-sheet content and an increased random coil content, but maintaining the trimeric quaternary structure. This conformational change in the MIF molecule enables binding of oxMIF-specific antibodies BaxB01 and BaxM159, which showed beneficial activity in animal models of inflammation and cancer. Crystal structure analysis of the MIF-derived EPCALCS peptide, bound in its oxMIF-like conformation by the Fab fragment of BaxB01, revealed that this peptide adopts a curved conformation, making the central thiol protein oxidoreductase motif competent to undergo disulfide shuffling. We conclude that redMIF might reflect a latent zymogenic form of MIF, and formation of oxMIF leads to a physiologically relevant, i.e., enzymatically active, state.

Macrophage Migration Inhibitory Factor (MIF)-Based Therapeutic Concepts in Atherosclerosis and Inflammation

“…Based on biochemical and immunochemical experiments, it was suggested that this antibody recognizes an oxidized form of MIF with the oxidoreductase motif of MIF trapped in an oxidized state, and that Cys-81 serves as a molecular redox switch between the latent reduced form of MIF and its oxidized state. 93,98,99 While the antibody was reported to detect an oxidized MIF species termed 'oxMIF' in tumour tissue from patients with colorectal, pancreatic, ovarian and lung cancer, 100 the claim that oxMIF is the pathophysiologically relevant MIF species appears speculative and convincing evidence that imalumab targets pathogenic oxMIF species is missing. 67,95 MIF-2/D-DT shares with MIF a pronounced pro-inflammatory activity profile and has been reported to promote endotoxaemia, adipose tissue inflammation and tumorigenesis similar to MIF.…”

Advances and Insights for Small Molecule Inhibition of Macrophage Migration Inhibitory Factor

“…Secondly, Cys-80 is believed to act as a “switch cysteine” for the conversion of redMIF to the disease-related isoform oxMIF. 30 Covalent modification of Cys-80 with a series of pro-oxidative reagents showed that while some precipitated the conversion to oxMIF, at least one covalent adduct remained in the reduced form. This finding might indicate that a covalent inhibitor may be capable of trapping the comparatively benign redMIF oxidation state.…”

Small-molecule inhibitors of macrophage migration inhibitory factor (MIF) as an emerging class of therapeutics for immune disorders

“…MIF, but not D-DT, has a CXXC motif that can be oxidized to an intramolecular disulfide-bond. Oxidised MIF has also been proposed as a biomarker for different diseases [28]. Other studies indicate that oxidized MIF is the disease-related conformational isoform that could be employed for diagnosis and therapy [29][30].…”