Apoptosis is commonly seen in multicellular organisms as a method to regulate the development of said organism. A similar, programmed cell death pathway has been identified in the unicellular organisms, yeast and P. falciparum. In yeast, the cell death pathway is initiated once a target protein is activated, which causes a downstream proteolytic cascade to activate. The target protein is Apaf-1 (apoptosis promoting factor 1), which binds to cytochrome c. Apaf-1 then triggers the formation of an apoptosome, which, when induced by ATP, turns on the other proteases downstream of Apaf-1. Recent studies suggest that P. falciparum might contain a similar cell death pathway to yeast. Our aim is to identify the players of the putative apoptosis pathway in the malaria parasite. The activation of the cell death pathway was tested using a fluorogenic substrate (z-VAD-AMC). The assay aims to demonstrate that z-VAD-AMC is cleaved once the yeast is exposed to increasing concentrations of cytochrome c. This assay was also used on P. falciparum, using the Pfcytochrome c as the initiator of the pathway. Plasmodium falciparum cytochrome c was expressed in electrocompetent E. coli C41 cells via electroporation of two plasmids, pMKL1 and pEC86, with the latter containing the cytochrome c maturation system. We will report on the expression and purification of the target protein, as well as the results of the assay.

Role of cytochrome c and its effects on the programmed cell death pathway of P. falciparum using yeast as a model organism

249th National Meeting (2015)

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