Pro-inflammatory activity of snake venom proteinases: i)expression of recombinant proteinases in a cell-free system; ii) generation of microvesicles by monocytes stimulated with native and recomb proteinases, and analysis of their interaction with end cells
Cells release different forms of extracellular vesicles, including structures called microvesicles (MVs) that are capable of altering the extracellular microenvironment. Despite the growing number of studies on MVs, their biogenesis, biological function, and the mechanisms that regulate the delivery of their contents are still poorly known. MVs are generated from various cell types such as endothelial cells, platelets, monocytes, tumor cells, and may be involved in physiological and pathological events by participating in cell-to-cell communication through interactions mediated by receptors or transfer of bioactive molecules, including membrane receptors, proteins, lipids, tRNAs, mRNAs, miRNAs and organelles. Toxins of viperid venoms induce an acute inflammatory response that contributes to the severity of symptoms observed upon envenomation, which possibly involves the release of MVs. However, the role of MVs in the pathological context of snake bite envenomation is not known. Therefore, one of the objectives of this project is to analyze the effect of MVs generated by monocytes activated by a metalloproteinase (bothropasin) and a serine proteinase (PA-BJ) from Bothrops jararaca venom, in native and recombinant forms, upon endothelial cells. The content of MVs will be assessed using proteomic and peptidomic approaches in order to understand how these MVs could affect the endothelium in the envenomation process. Moreover, in this project we also aim to set up the production of recombinant toxins using a cell-free expression system derived from Escherichia coli, for the first time in Brazil. The success in establishing this project at Butantan Institute will allow the implementation of a new platform for the production of recombinant proteins of academic and biotechnological applications.
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