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Macrophage migration inhibitory factor (MIF) is rendered enzymatically inactive by myeloperoxidase-derived oxidants
Nina Dickerhof1, Lisa Schindler2, Jürgen Bernhagen2
1Centre for Free Radical Research, Department of Pathology, University of Otago Christchurch, Christchurch, New Zealand.
Abstract:
Macrophage migration inhibitory factor (MIF) is an important player in the regulation of the inflammatory response. Elevated plasma MIF is found in sepsis, arthritis, cystic fibrosis and atherosclerosis. Immunomodulatory activities of MIF include the ability to promote survival and recruitment of inflammatory cells and to amplify pro-inflammatory cytokine production. MIF has an unusual nucleophilic N-terminal proline with catalytic tautomerase activity. It remains unclear whether tautomerase activity is required for MIF function, but small molecules that inhibit tautomerase activity also inhibit the pro-inflammatory activities of MIF. A prominent feature of the acute inflammatory response is neutrophil activation and production of reactive oxygen species, including myeloperoxidase (MPO)-derived hypochlorous acid and hypothiocyanous acid. We hypothesized that MPO-derived oxidants would oxidize the N-terminal proline of MIF and alter its biological activity. MIF was exposed to hypochlorous acid and hypothiocyanous acid and the oxidative modifications on MIF were examined by LC-MS/MS. Imine formation and carbamylation was observed on the N-terminal proline in response to MPO-dependent generation of hypochlorous and hypothiocyanous acid, respectively. These modifications led to a complete loss of tautomerase activity. However, modified MIF still increased CXCL-8/IL-8 production by peripheral blood mononuclear cells (PBMCs) and blocked neutrophil apoptosis, indicating that tautomerase activity is not essential for these biological functions. Pre-treatment of MIF with hypochlorous acid protected the protein from covalent modification by the MIF inhibitor 4-iodo-6-phenylpyrimidine (4-IPP). Therefore, oxidant generation at inflammatory sites may protect MIF from inactivation by more disruptive electrophiles, including drugs designed to target the tautomerase activity of MIF.
Insights
Oxidative modification of macrophage migration inhibitory factor (MIF) by reactive oxygen species at inflammatory sites inactivates its tautomerase activity but preserves its pro-inflammatory functions. This suggests MIF oxidant modification may protect it from drug inactivation.
Area of Science:
- Biochemistry
- Immunology
- Molecular Biology
Background:
- Macrophage migration inhibitory factor (MIF) is a key regulator of inflammation, implicated in diseases like sepsis and arthritis.
- MIF possesses unique N-terminal proline tautomerase activity, crucial for its pro-inflammatory functions, though its necessity is debated.
- Reactive oxygen species (ROS), such as hypochlorous acid and hypothiocyanous acid generated by myeloperoxidase (MPO), are prominent during acute inflammation.
Purpose of the Study:
- To investigate the hypothesis that MPO-derived oxidants modify the N-terminal proline of MIF, altering its biological activity.
- To determine if MIF's tautomerase activity is essential for its immunomodulatory functions.
- To explore the implications of MIF oxidation in the context of inflammatory drug development.
Main Methods:
- Exposure of MIF to hypochlorous acid and hypothiocyanous acid.
- Analysis of oxidative modifications using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
- Assessment of modified MIF's tautomerase activity and biological functions, including cytokine production and neutrophil apoptosis, in peripheral blood mononuclear cells (PBMCs).
Main Results:
- Oxidation of MIF's N-terminal proline by hypochlorous acid and hypothiocyanous acid resulted in imine formation and carbamylation, respectively.
- These modifications completely abolished MIF's tautomerase activity.
- Despite loss of tautomerase activity, modified MIF retained its ability to enhance CXCL-8/IL-8 production and inhibit neutrophil apoptosis.
Conclusions:
- MIF's tautomerase activity is not essential for its pro-inflammatory functions, such as promoting cytokine production and blocking neutrophil apoptosis.
- Oxidant generation at inflammatory sites can modify MIF, potentially protecting it from inactivation by electrophilic inhibitors targeting its tautomerase activity.
- These findings have implications for understanding MIF's role in inflammation and for designing targeted therapies.
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