| Two novel families of caspase-like proteins, metacaspases and paracaspases, have been discovered but have remained poorly characterized. Metacaspase type-1 (Acmcp) has been identified in Acanthamoeba castellanii, which is an opportunistic protozoan parasite that causes several diseases in humans. On the other hand, paracaspase (Ddpcp) has been found in the protozoan Dictyostelium discoideum. D. discoideum is well-researched model organism that is appropriate for studying these caspase-like proteins. In this study, we investigated the localization and molecular function of metacaspase and paracaspase proteins by over-expressing them in D. discoideum. Separate cell lines over-expressing a GFP-tagged version of the full length Acmcp gene (GFP-Acmcp), a deletion proline region mutant version of the Acmcp gene (GFP-Acmcp-dpr), a GFP-tagged version of the full length Ddpcp gene (GFP-pcp) and a Ddpcp knockout version, have been expressed in D. discoideum . Experimental results from microscopic analysis of cells expressing Acmcp and Ddpcp proteins have revealed that both of the GFP-tagged caspases localize and function with the contractile vacuolar (CV) system in Dictyostelium including in the process of the fusion of the CV with the plasma membrane. A yeast two- hybrid system was used to investigate if Ddpcp and Acmcp interact with the other contractile vacuolar proteins (Calmodulin, Rab D, Rab 11 and vacuolar proton pump ATPase). This assay identified that Acmcp have a weak interaction with the vacuolar proteins while pcp showed strong interaction with the vacuolar proton pump protein. A functional analysis suggests that the mutant cells showed differences in growth rate, significant increases in fluid internalization rate, and significant increases in phagocytosis rate. Furthermore, the severe defect in development phenomena in cells overexpressing GFP-pcp and GFP-Acmcp-dpr suggests that Ddpcp and Acmcp have an essential role in binding these proteins with other partners to maintain these processes. Taken together, our findings suggest for the first time that the Ddpcp and Acmcp, caspases-like proteins, are associated with the contractile vacuolar system. Additionally, the functional analysis of Ddpcp and Acmcp in Dictyostelium demonstrated that these proteins have essential roles in cell osmoregulation, endocytosis, phagocytosis, cell aggregation, and development, which contribute to their attractiveness as possible targets for treatment therapies. |
