Production of yeast metacaspases with N-terminal depletion and containing site-directed mutations and biochemical characterization of these enzymes

Abstract

The present project is a continuation of the previous project, biochemical characterization of caspase-like cells involved in the simple eukaryotic cell cycle (FAPESP - 2015 / 11190-0), during the execution of the project we observed that the metacaspases of yeasts belonging to the group I of this class of enzymes, present in the N-terminal portion a sequence rich in glutamine and asparagine that promotes the protein aggregation of these molecules, making their solubility impossible and consequently impeding their biochemical characterization. For this reason, this research project aims at the cloning of these enzymes with the deletion of the N-terminal region. The metacaspases undergo a self-processing to become catalytically active, and require the calcium ion as an enzymatic cofactor both for its self-processing and to perform its enzymatic activity, for that reason we intend to carry out the production of these enzymes containing site mutations directed at key residues for enzymatic activity and in the sites of their self-processing, to try to elucidate the importance of this self-processing for the regulation of the activity of these proteases. (AU)