Characterization of CAP5.5 and CAP5.5V, atypical calpains in Trypanosoma brucei

Calpains are a group of calcium-dependent cysteine proteases with roles ranging from cytoskeletal remodelling to signal transduction. In Trypanosoma brucei, the causal agent of African sleeping sickness, an unusually large number of calpain- like proteins exist, whose functions remain unknown. Recently, the procyclic form- specific cytoskeleton associated protein 5.5 (CAP5.5) and its bloodstream-specific paralogous variant, CAP5.5V were discovered to be essential for the proper morphogenesis of the parasite. However, precise functional roles of these proteins remain unknown. In this MSc thesis, I employed a two-pronged approach combining cell biology studies of CAP5.5 and CAP5.5V in T. brucei with in vitro assays of recombinant calpains expressed in Escherichia coli to further characterize CAP5.5 and CAP5.5V. To resolve whether the function of CAP5.5 and CAP5.5V is dependent upon proteolytic activity, I expressed and purified recombinant CAP5.5V’s catalytic domain. I demonstrated that this domain did not have any detectable autolytic activity or activity against universal protease substrates such as casein. Ectopic expression of CAP5.5::YFP::Ty in procyclic trypanosomes led to nuclear mis-positioning among other morphological defects. Additionally, I demonstrated a cell cycle dependency on incorporation of CAP5.5::YFP::Ty into the cytoskeleton and corroborate previous data that suggest that CAP5.5 is built in from the dynamic, posterior end of the cell. Investigation of the molecular determinants in CAP5.5’s sequence such as motifs or domains suggested that CAP5.5’s domain IV may be the domain mediating association with the sub-pellicular cytoskeleton.This thesis is not currently available in ORA