Interaction Crotoxin and Macrophages: Study of the participation of Formyl Peptide Receptor and Lipid Domains in Mechanisms Involved in the Molecular Toxin-Membrane Recognition Process

Abstract

Several studies have shown that Crotoxin (CTX), the main toxin of South American rattlesnake (Crotalus durissus terrificus) formed by the association of subunits CA and CB, exhibits in vitro and in vivo, antitumor, anti-inflammatory, antiviral and immunomodulatory activities. With regard to modulating activities, CTX stimulates in particular, macrophage metabolism and the production of cytokines, lipoxin A4 and analogous 15-epi-LXA4 secretion by these cells, leading to inhibition of events involved in tumor progression and angiogenesis. These CTX actions are blocked in their entirety by Boc-2 selective antagonist formyl peptide receptors. This finding highlights the importance of these receptors to the immunomodulatory activities of CTX. However, despite this evidence, it is not known yet how the CTX induces their actions in macrophages, as well as how these receptors are involved. Therefore, characterizing the process of molecular recognition of CTX in macrophages is crucial to understand how this toxin can lead to different activities on metabolism and function of these cells. The aim of this project is to evaluate the interaction of CTX complex structures of the cell membrane of macrophages, such as formyl peptide receptor and lipid domains involved with the membrane structure and pore formation, essential for the transduction of signals and functions cells. For this: 1) Testing of biological activities, in 2D and 3D models, in order to establish the participation of the FPR, the effect of CTX on the functional activity of macrophages. At this stage, it will evaluate the expression of the formyl peptide receptor in THP-1 cell, a cell of monocytic lineage by way of silencing. The muting is accomplished by means of electroporation technique with nucleofection technology. THP-1 silenced or not, they are incubated with CTX-labeled fluorescent probe and evaluated in Time Lapse assay. After silencing the functional assays such as phagocytosis and production and release of H2O2 and NO, respectively, are determined; 2) In tests of structural term phase, to evaluate CTX-membrane interaction, liposomes or synthetic vesicle will be used, formed from synthetic lipids, neutral or charged, which are more abundant in the cell membrane of macrophages will be used in assays CTX-membrane interactions. These tests will assess, through Electron Paramagnetic Resonance (EPR) and Differential Scanning Calorimetry (DSC), which types of interactions with the membrane CTX is able to perform. Considering the important activity of CB (Phospholipase A2 - PLA2) on the phospholipid membranes are also used CA and CB subunits dissociated, in order to compare the activities of CTX complex. Importantly, these physical techniques are well established and frequently applied in chemistry, biochemistry, cell biology, biotechnology, pharmacology, and recently in nanoscience. (AU)