Detection of Caspase Activation in Cells by Ricin Using Fluorescence Resonance Energy Transfer (FRET)
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Ricin is naturally occurring, type-2 ribosome inactivating protein produced by castor seeds (Ricinius communis). Ricin is considered a potential bioterrorism weapon due to its extreme toxicity and the relative ease by which it can be obtained from castor seeds. The ability to detect biologically active ricin is important for the FDA mission. The catalytic A subunit of ricin deactivates up to 2000 ribosomes per minute causing the termination of protein synthesis. The protein synthesis blockade initiates signaling pathways leading to apoptotic cell death via activation of caspase 3 and 8. The detection method studied in this project used FRET (Fluorescence Resonance Energy Transfer) to detect caspase activation induced by exposure to ricin. FRET is a phenomenon in which energy absorbed by one fluorescent molecule is transferred to another fluorescent molecule. The fluorescent emission of cyan fluorescent protein (CFP) overlaps the excitation of yellow fluorescent protein (YFP), allowing FRET to occur when they are in close proximity. A recombinant expression vector coupling CFP to YFP via a short peptide linker containing caspase 3 and 8 target sequences was introduced into MB231 breast carcinoma cells and RAW264.7 macrophage cells. Exposure of these cells to ricin activates caspase which diminishes the FRET signal. In this study, we evaluated the time course for diminished FRET intensity caused by exposure to ricin and found that a significant change can be detected after 3.5 hours in RAW 264.7 CFP-YFP cells and after 7.5 hours in MB231 CFP-YFP cells, which is much faster compared to the standard 48 hour cytotoxicity assay.