Inventor: Dr. Entsar Jabbar Saheb/Department of Biology, College of science, University of Baghdad
Dictyostelium discoideum amoeba represents an important system to study the function of various genes because it contains many genes that are similar to higher eukaryotes. Novel relatives to caspases proteins have been discovered including paracaspases that have been found in protozoan D. discoideum. Since its discovery, not much is known about the molecular function of paracaspase proteins. The function of paracaspases protein at the molecular level is an important area of study that would provide adequate information about its functional role in the most complex organisms, including humans. In addition, such studies provide information about the possibility of its use as a treatment against several parasitic amoeba. In this study, two separate mutant cell lines were first analysed using Green Flourecent Protein (GFP): one cell line was an over-expressed GFP-tagged Pcp (GFP-Pcp), while the other cell line was a pcp-null (pcp-). Microscopic analysis of cells expressing GFP-Pcp revealed that Pcp was associated with the contractile vacuole membrane consisting of bladder-like vacuoles and has a great role in their composition. In addition, the functional analysis showed that the GFP-Pcp-over-expressing cells were susceptible to osmotic stress and contained more abnormally swollen contractile vacuole. Furthermore, yeast two-hybrid system was used to identify whether Pcp protein interact with the other proteins that found in the contractile vacoul such as Calmodulin, Rab D, Rab 11, vacuolar proton pump ATPase (VatM). The results showed that pcp protein associated with VatM. In addition, the functional analysis made clear that the mutant cells showed differences in growth rate and a significant increase in the rate of absorption of fluids, and a significant increase in the rate of phagocytosis. Moreover, it was observed that the GFP-Pcp cells that produce greater amounts of Pcp was not able to complete their life cycle even in the early rounds. In addition, these cells were not capable of sticking or assembly to move to subsequent eccentric in their life cycle, while the opposite was seen in cells that has lost protein Pcp (pcp-). From these results it can be concluded that this work gives evidence for the first time that eukaryotic paracaspase to be associated with both localization in and regulation of the contractile vacuolar system, an organelle critical for maintaining the normal morphology of the cell. Also, this study propose that Pcp is a regulated protein involved in cellular functions including signal relay pathways that are essential for cell aggregation and development. Thus, Pcp protein is a candidate drug target against the amoebic parasitic infection by prevent development to their infective stages that are currently resistant to various types of treatment.