Caspases are cysteine proteases in animals that play an important role in programmed cell death regulation and execution. Caspases are absent in non animal eucaryotes; however, they contain structural homologues of caspases, metacaspases. These can be divided into three types that can be differentiated based on their domain architectures. Their function is yet not known, although it is likely linked to oxidative stress responses.
In this work, we focused on CrMCA-I, the only type I metacaspase from the unicellular green alga Chlamydomonas reinhardtii. When expressed in E. coli, it aggregated in the insoluble fraction. However, removal of the N-terminal region and the 360 loop (CrMCA-I_CL) resulted in expression of soluble and proteolytically active protease. Since this loop is characteristic of type I metacaspases from organisms of the Viridiplantae clade, we also tried to express two sequences encoding metacaspases from Arabidopsis thaliana, AtMCA-Ia and AtMCA-Ib. However, we were not successful in their expression.
We measured the activity of the isolated CrMCA-I_CL at different concentrations of calcium ions and different pH values. CrMCA-I_CL was shown to be inactive in the absence of Ca2+ ions and reached its maximum activity at low millimolar Ca2+ concentrations. Its pH optimum was at neutral values.
We also tried to express non-modified CrMCA-I homologously in C. reinhardtii, but we encountered problems with establishing stable colonies after transformation. While colonies did appear on solid medium with antibiotic, no growth could be observed in liquid media.
To asses if presence of metacaspases during oxidative stress response could be monitored using anti-metacaspase antibodies, we induced oxidative stress in the wild type C. reinhardtii and in CrMCA-I insertion mutant CrMCA-I::AphVII. Since no metacaspase could be detected, other methods will have to be used in the future for monitoring the presence and activity of metacaspases in this organism.
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