Year: 2008
Type: dissertation
Abstract: The cytokine macrophage migration inhibitory factor (MIF) is a key mediator of the innate and adaptive immune system and plays a critical role in many inflammatory diseases. MIF is required to combat serious infections; however, high-level production of MIF has been linked to a severe outcome of many diseases including adult respiratory distress syndrome, septic shock, rheumatoid arthritis, atherosclerosis and cancer. Over the last years, MIF has also been ascribed important functions in the host defense against several parasitic infections. Consequently, anti-MIF therapies have been suggested as a potential therapeutic approach for treating MIF-related diseases. Unique among cytokines, MIF possesses an enzymatic activity that is evolutionarily conserved. Herein, 4-iodo-6-phenylpyrimidine (4-IPP), a MIF inhibitor which is ~ 5-10 times more potent in blocking MIF-dependent catalysis than other prototypical MIF inhibitors is described. Crystallographic studies reveal 4-IPP to serve as a suicide substrate for MIF, resulting in the covalent modification of the catalytically active N-terminal proline and loss of function of MIF-induced monocyte migration. Additionally, two parasitic orthologs of MIF, which are produced by the obligate intracellular parasites, Leishmania major (LmMIF) and Plasmodium falciparum (PfMIF) were studied. An interest in the structure and function of MIF orthologs from parasites has emerged recently as they might have relevant functions in corrupting the host MIF’s induced immune defense mechanisms. By co-evolving with the immune system, parasitic organisms have evolved specialized strategies to circumvent the host’s immune defense mechanisms to increase their own chances of survival. To assess whether LmMIF and PfMIF have the potential to disrupt immunological pathways of the host’s immune system, the parasitic proteins were recombinantly produced and purified. LmMIF and PfMIF show significant binding interaction with the human MIF receptor, CD74 (Kd ~ 28 nM), and like its mammalian counterpart, the recombinant LmMIF protein is internalized by macrophages, induces monocyte cell migration, ERK1/2 MAP kinase activation, inhibits the activation-induced apoptosis of macrophages and binds the Golgi-associated tethering protein p115. The Leishmania MIF protein shows significant structural homology with human MIF as revealed by a high-resolution x-ray crystal structure (1.03 Å). Significant differences between the two proteins in the N-terminal tautomerization site are evident, and evidence for the selective, species-specific inhibition of MIF by small-molecule antagonists that target this site is provided. Finally, this thesis was aimed to further elucidate the molecular basis of MIF’s chemokine-like functions which were recently shown to be mediated through interaction to the chemokine receptors CXCR4 and CXCR2. However, the molecular details of this interaction have not yet been determined. Herein, a peptide derived from the N-terminal extracellular region of the CXCR4 receptor as a site of interaction with human MIF and Leishmania major MIF is described. From a competitive binding assay and 1H15N chemical shift perturbation studies, a direct binding interaction between MIF and the N-terminal 27 residues of CXCR4 is shown. Titration studies using steady-state fluorescence spectroscopy resulted in a dissociation constant of 3.1 µM. Notably, LmMIF/MIF-triggered monocyte chemotaxis activity is ablated by this N-terminal CXCR4 peptide. The present study not only provides a crystallographic characterization of the prototypic MIF inhibitor 4-IPP for a potential treatment of MIF-related autoimmune and inflammatory diseases, but also unfolds possible new pathways of parasitic MIF-orthologs to interfere with the host immune system. The study of the molecular mode of interaction of MIF and its binding partners will provide new opportunities to block these interactions specifically, and thus may aid in the design of new drugs targeting MIF-related diseases and immune evasive strategies of parasites.
Source: RWTH Publications (RWTH Aachen)
Authors: Swen Zierow, Jürgen Bernhagen
Language: English
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W651397705