Interaction of macrophage migration inhibitory factor with ceruloplasmin: role of labile copper ions
Valeria A Kostevich1,2, Alexey V Sokolov3,4,5, Natalia A Grudinina1
1Institute for Experimental Medicine, Pavlov Street 12, Saint Petersburg, 197376, Russia.
Abstract:
Macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, is a target for pharmacological treatment of sepsis and malignant tumors. Inhibition of tautomerase activity of MIF in reaction with p-hydroxyphenylpyruvate (HPP) was observed in the presence of ceruloplasmin (CP), a copper-containing plasma protein. Binding labile copper ions to CP (CP+Cu(II)) is a prerequisite for MIF inhibiting. CP+Cu(II) is shown to be an uncompetitive inhibitor of MIF (Ki ~ 37 nM), which suggests formation of a complex 'MIF-HPP-CP-Cu(II)'. Filtration of CP+Cu(II) on a column with Chelex-100, otherwise the presence of high concentrations of histidine, cysteine or methionine abrogated the inhibitory effect of CP. Adding salts of Co(II) and Ni(II) that replace copper ions in the labile sites prevented the inhibitory effect of CP+Cu(II). Limited proteolysis of CP by thrombin diminished its oxidase activity in reaction with p-phenylenediamine, but endowed it with the capacity of inhibiting MIF. Covalent modification of MIF by phenylmethylsulfonyl fluoride (PMSF) resulted in binding of MIF-PMSF to CP immobilized on CM5 chip, the dissociation constant being 4.2 μM. In D-galactosamine-sensitized mice CP+Cu(II) increased the LPS-induced lethality from 54 to 100%, while administration of antibodies against MIF prevented the lethal effect. The enhancement by CP+Cu(II) of the pro-inflammatory signal of MIF is discussed.
Insights
Ceruloplasmin bound to copper ions inhibits macrophage migration inhibitory factor (MIF) activity. This interaction, crucial for inflammatory responses, was blocked by chelating agents and specific amino acids.
Area of Science:
- Biochemistry
- Immunology
- Pharmacology
Background:
- Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine targeted for treating sepsis and tumors.
- Ceruloplasmin (CP), a copper-binding protein, influences biological processes.
- Understanding MIF and CP interactions is key to developing novel therapeutics.
Purpose of the Study:
- To investigate the inhibitory effect of ceruloplasmin (CP) on the tautomerase activity of macrophage migration inhibitory factor (MIF).
- To elucidate the role of copper ions in the CP-mediated inhibition of MIF.
- To explore the potential of this interaction in modulating inflammatory conditions.
Main Methods:
- Enzyme inhibition assays measuring MIF tautomerase activity in the presence of CP and copper ions (CP+Cu(II)).
- Inhibition studies using chelating agents (Chelex-100) and amino acids (histidine, cysteine, methionine).
- In vivo studies using D-galactosamine-sensitized mice to assess the effect of CP+Cu(II) on LPS-induced lethality.
Main Results:
- CP+Cu(II) acted as an uncompetitive inhibitor of MIF with a Ki of approximately 37 nM, suggesting a ternary complex formation.
- The inhibitory effect was abrogated by chelating labile copper ions or by the presence of histidine, cysteine, or methionine.
- CP+Cu(II) significantly increased LPS-induced lethality in mice, an effect counteracted by anti-MIF antibodies.
Conclusions:
- Ceruloplasmin, when bound to labile copper ions, effectively inhibits MIF tautomerase activity.
- This interaction enhances the pro-inflammatory effects of MIF in vivo, increasing lethality in a sepsis model.
- The findings highlight a novel mechanism of MIF regulation with implications for inflammatory disease therapies.
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