whereRU: Aresty Poster 4 - Studying the biological activity of ricin in mammalian cells by Aresty Posters 2009

Use of site-directed mutagenesis to study the biological activity of ricin in mammalian cells
Adip Pillai, Amanda Jetzt and Wendie Cohick

Ricin is a ribosome-inactivating cytotoxic protein easily derived from the castor bean plant and considered to be a possible bioterror agent classified as a level B biothreat. Ricin is a holotoxin composed of an A chain (RTA) and a B chain (RTB) of which the RTA is the active chain. RTA is known to induce apoptosis, however, the link between protein synthesis inhibition and apoptosis has not been clearly established. Previous experiments in yeast have shown that protein synthesis inhibition is not necessarily sufficient for cytotoxicity. It is unknown whether this finding can be extrapolated to mammalian cells, which have distinct apoptotic pathways not found in yeast. We hypothesize that mutants of RTA that have had their active sites altered so that they cannot depurinate the ribosome but can still bind it will retain the ability to activate apoptosis even though they do not inhibit protein synthesis. This hypothesis will be tested by creating an RTA double mutant using site-directed mutagenesis. This mutant will be transfected into mammalian cells and then a series of biological assays will be conducted to test for depurination, apoptosis, and protein synthesis inhibition. The results will show what relationship, if any, exists between apoptosis and protein synthesis inhibition through ribosome depurination. This experiment will allow a better understanding of the mechanisms of ricin allowing for possible treatments to be developed.