Metacaspases are cysteine proteases, which are structural homologues of caspases and therefore belong to the C14 family of the CD clan. They can be found in various organisms, including bacteria, archaea, algae, and unicellular eukaryotes, but not in metazoa. In addition to caspase-specific domains p20 and p10, they can contain other regions, based on which they are classified as type I, type II and type III. Unlike caspases, they preferentially cleave substrates with basic amino acid residues in the P1 position. Additionally, most metacaspases require calcium for activation, while some also undergo specific autoprocessing. This is particularly true of type II metacaspases, which have been shown to require cleavage after a conserved basic amino acid residue in the linker region for activation.
The aim of this thesis was to study the structure-function relationship of
metacaspase-specific structural elements and to discover their role in activity and activation. We performed an in silico analysis of various amino acid sequences of type I and type II algal metacaspases and identified conserved amino acid residues. Based on the analysis, we identified specific residues in the model metacaspases CrMCA-I and CrMCA-II from the algae Chlamydomonas reinhardtii, which could play an important role in their function. Furthermore, we constructed mutants by introducing point mutations. For CrMCA-I we mutated amino acids, involved in specificity and activation, while for CrMCA-II we selected residues, that could represent potential cleavage sites during activation. We isolated recombinant CrMCA-I_CL and its mutants. Next, we compared the activity of the isolated enzymes towards protein and peptide substrates and determined the kinetic parameters for the cleavage of selected synthetic substrates. We discovered that while all the mutants can cleave large protein substrates, their activity towards peptide substrates is lower in comparison to the initial protein. Additionally, we isolated and purified CrMCA-II, but were unsuccessful with the isolation of its mutants. We analysed the rate of autoprocessing of CrMCA-II at different temperatures and calcium concentrations. Our results demonstrate that in the presence of Ca2+ and 4 °C, CrMCA-II undergoes immediate autoprocessing which yields two fragments. With longer incubation times and higher temperatures, there is an increase in cleavage rate, which finally leads to a complete degradation of the protein.
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