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Characterization of the Macrophage migration Inhibitory Factor of Leishmania major (LmjMIF2): contribuition of the oligomeric state for structure and function

Grant number: 16/10331-2
Support type:Regular Research Grants
Duration: September 01, 2016 - November 30, 2018
Field of knowledge:Biological Sciences - Biochemistry - Chemistry of Macromolecules
Principal Investigator:Arthur Henrique Cavalcante de Oliveira
Grantee:Arthur Henrique Cavalcante de Oliveira
Home Institution: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

The macrophage migration inhibitory factor (MIF), originally described as a cytokine of human T cells, inhibits the migration of macrophages. MIF is considered an important factor in the control of infection by parasites. Resistant mice deficient in MIF-/ - became more susceptible to infection by L. major and Trypanosoma cruzi. The description in the literature of the homologous MIF secretion in parasites suggests a parasite immune response modulation by interaction with the macrophage receptor (CD74). Recently, it was shown that one of the two MIFs from L. major (LmjMIF1) acts inhibiting apoptosis, indicating that LmMIF contributes to the macrophages infected survival. Thus, the objective of this project is to investigate the role of intersubunit interactions in the structure stability and in the biological function of the L. major MIF (LmjMIF2). It is intended to achieve this objective by biophysical and biochemical characterization of the C-terminal mutants containing a single tryptophan in the structure.Mutant sequences of LmMIF2 with C-terminal deletions and containing a single tryptophan will be produced by PCR reactions and cloned. The proteins will be expressed in E. coli and purified by affinity chromatography. Solutions studies as circular dichroism and IFTE (intrinsic fluorescence tryptophan emission) using thermal variation and denaturing agents will allow to map the profile of the structural changes of regions containing tryptophan (the core protein and the C-terminal tail of the molecule). The monitoring of these conformational changes in the tertiary structure of these mutants, will indicate the importance of these regions for maintaining the folded structure of LmjMIF2. The oligomeric state will be evaluated by gel filtration, Small Angle X-ray Scattering (SAXS) and native polyacrylamide gels. Thus, the mutants containing the tryptophan in the oligomeric interface , coupling with spectroscopic studies in solution, can be contribute to indicate the oligomeric state of the LmMIF2 and if this structure interacts with macrophage receptor. The functional activity will be evaluated by migration inhibition of J774 macrophages and will indicate the role of oligomerization state of LmMIF2 in the host cell/parasite interactions. Further, the obtainment promastigote forms overexpressing LmMIF2 and mutants will be obtained and used in the footpad lesion and the macrophages invasion analysis. So with these results we intend to correlate the role of the C-terminal region in the LmMIF2 structure maintenance and functional activity that occur in the host-parasite interaction.The study of the structure solution of mutants, especially the C-terminal tail will help to characterize the maintenance of quaternary structure in solution relating it to the functional activity in the macrophage receptor interaction. These studies will allow the identification of regions of Leishmania homolog of MIF2 different of the human MIF, which may be essential for activity during the parasite's life cycle. These regions may, in future, binding sites for new drugs to combat development of the parasite (AU)