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Direct link between cytokine activity and a catalytic site for macrophage migration inhibitory factor
1Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510, USA.
Abstract:
Macrophage migration inhibitory factor (MIF) is a secreted protein that activates macrophages, neutrophils and T cells, and is implicated in sepsis, adult respiratory distress syndrome and rheumatoid arthritis. The mechanism of MIF function, however, is unknown. The three-dimensional structure of MIF is unlike that of any other cytokine, but bears striking resemblance to three microbial enzymes, two of which possess an N-terminal proline that serves as a catalytic base. Human MIF also possesses an N-terminal proline (Pro-1) that is invariant among all known homologues. Multiple sequence alignment of these MIF homologues reveals additional invariant residues that span the entire polypeptide but are in close proximity to the N-terminal proline in the folded protein. We find that p-hydroxyphenylpyruvate, a catalytic substrate of MIF, binds to the N-terminal region and interacts with Pro-1. Mutation of Pro-1 to a glycine substantially reduces the catalytic and cytokine activity of MIF. We suggest that the underlying biological activity of MIF may be based on an enzymatic reaction. The identification of the active site should facilitate the development of structure-based inhibitors.
Insights
Macrophage migration inhibitory factor (MIF) has a structure similar to microbial enzymes. Proline at position 1 is crucial for MIF
Area of Science:
- Biochemistry
- Immunology
- Structural Biology
Background:
- Macrophage migration inhibitory factor (MIF) is a key secreted protein involved in immune cell activation.
- MIF plays a role in inflammatory conditions such as sepsis, ARDS, and rheumatoid arthritis.
- The precise mechanism underlying MIF's biological functions remains largely unknown.
Purpose of the Study:
- To elucidate the functional mechanism of Macrophage migration inhibitory factor (MIF).
- To investigate the role of the N-terminal proline residue (Pro-1) in MIF activity.
- To identify the active site of MIF for potential therapeutic target development.
Main Methods:
- Comparative analysis of MIF structure with microbial enzymes.
- Site-directed mutagenesis of the N-terminal proline (Pro-1) residue.
- Biochemical assays to assess catalytic and cytokine activity.
- Analysis of substrate binding to the N-terminal region.
Main Results:
- MIF shares structural similarities with microbial enzymes, particularly at the N-terminus.
- The N-terminal proline (Pro-1) is invariant across MIF homologues and interacts with a substrate.
- Mutation of Pro-1 significantly diminishes both catalytic and cytokine activities of MIF.
Conclusions:
- The biological activity of MIF is likely based on an enzymatic reaction.
- The N-terminal proline (Pro-1) is essential for MIF's catalytic and immune-modulating functions.
- Identification of the MIF active site opens avenues for structure-based inhibitor design.