Site-directed Mutations in the Active-Site Oligomeric Interface Regions of the macrophage migration inhibitory factor of Leishmania major (LmjMIF2)

• Grant number: 09/04272-0
• Support Opportunities: Scholarships in Brazil - Master
• Effective date (Start): September 01, 2009
• Effective date (End): April 30, 2010
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Arthur Henrique Cavalcante de Oliveira
• Grantee: Elise Marques Freire Cunha
• Host 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. The objective of this project is the structural and functional investigation of the factor inhibiting migration of macrophages (MIF) of L. major using mutants in the active site and in the oligomer interface regions to assess the importance of these regions in the biological activity of LmjMIF. The selection of the residues in the structure of the MIF was based on the evaluation of the role of the enzyme activity and oligomer state in the biological activity. The mutants, K33A and G67A, were described as important for the tautomerization activity by to stabilize the substrate in the active site of mammalian MIF.All MIF described structures showed trimeric forms in the crystal, but the majority was detected as dimers in solution, including recombinant LmjMIF2 evaluated in our laboratory (Aux. Regular 07/06755-2). Thus, site-directed mutation of tryptophan residues and interface region, coupled with spectroscopic studies in solution can contribute to a response about the quaternary structure of MIF2 of L. major. Mutations in the oligomer interface, C-terminal loop (N109P and del103-113), will allow to evaluate the role of these residues and of the oligomer structure for biological activity. These mutants will be analyzed by gel filtration to indicate the oligomer state of the mutants. The mutants of tryptophans (W66L, W108F) will be used primarily for studies of the intrinsic tryptophan fluorescence emission (IFTE) and circular dichroism ring for evaluation of secondary, tertiary and quaternary mutant structures. Using fluorescence, will be evaluated and intrinsic anisotropy and fluorescence supression, which report on the suppression of access and movement of the side chain of tryptophans, allowing suggestions about tertiary and quaternary structures of MIF mutants. Thermal and agent denaturation studies using pH changes will be made by circular dichroism for secondary and tertiary structure analisys of these mutants, indicating the conformations changes in the active site and C-terminal regions during the MIF oligomerization. The analysis of mutants of the active site and oligomer interface regions will be correlated with the macrophages inhibitory migration activity, previously established in this laboratory, using LmjMIF2 recombinant. Thus, the interactions between mutant proteins and macrophages will evaluate the role of enzyme activity and quaternary structure of the biological activity of LmjMIF2.

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

CUNHA, Elise Marques Freire. Site-directed mutations in the regions of the active site and the interface oligomeric analysis of the Macrophage Migration Inhibitory Factor of Leishmania major (LmMIF2). 2011. Master's Dissertation - Universidade de São Paulo (USP). Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (PCARP/BC) Ribeirão Preto.