Programmed cell death plays a vital role in growth, development and maintenance of a multicellular organism. The process is crucial in tissue size retention, embryonal development, limb formation, elimination of damaged and infected cells and in proper functioning of the immune system. Apoptosis, together with necrosis and autophagy, presents the main type of programmed cell death. A crucial role in apoptosis is played by cysteine proteases termed caspases. Programmed cell death has an important role not only in animals but also in plant development and interaction with the environment. Key features distinguish plant programmed cell death from the one in animals. Plant cell wall hinders the formation of apoptotic bodies, the absence of phagocytes prevents the elimination of cell remains. Last but not least there are no coding sequences for apoptosis regulators, Bcl-2 and Bax families and caspases. Plant genomes, however, contain genes encoding metacaspases. Metacaspases, like their structural homologues caspases, possess catalytic dyad composed of cysteine and histidine. The difference between these two members of the C14 family is the substrate specificity. Caspases cleave after negatively charged amino acid residues as opposed to metacaspases, which preferably cleave after the positively charged amino acid residues. Furthermore, metacaspases require calcium ions for their activation, whereas caspases are known for their activation through dimerisation. Regarding the organisation of two caspase-like domains, p20 and p10, we distinguish three types of metacaspases. Type I metacaspases possess the same architecture as caspases, p10 being on the C-terminus of the p20 domain. Type I metacaspases can also contain a prodomain, which can be found in front of p20 and is cleaved after proteolytic processing. Type II is known for its long linker separating the two domains. Lastly, type III metacaspases have a different arrangement of the domains, p10 being at the N-terminus of p20.
The aim of this thesis was to prepare and isolate recombinant metacaspases of all three types and to determine their proteolytic properties. We used metacaspase 1 and 2 (GtMC1, GtMC2) from an algal organism Guillardia theta as representatives of type I and III, respectively, and metacaspase 2 from a model algal organism Chlamydomonas reinhardtii (CrMC2) as an example of type II metacaspases. E. coli expression system was used to express proteins. Proteolytic properties were determined using two substrates that are efficiently cleaved by metacaspases, FITC-casein and Z-FR-AMC. The data collected showed that all three metacaspases required millimolar concentrations of calcium ions for their activation. When it comes to pH dependency the metacaspases are not so unified. Two proteases from G. theta exhibited their optimal activity in acidic (pH 6,0), whereas metacaspase from C. reinhardtii prefered slightly basic conditions (pH 8,5).
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