The cellular process of programmed cell death, or apoptosis, is critical in homeostasis and development. In addition it's misfunction is implicated in an array of disease states from cancer to neurodegeration, making it an attractive pathway for drug targeting. A family of proteases, known as caspases, plays a central role in the apoptotic cascade resulting in the ultimate destruction of the cell. We report a genetically encoded dark-to-bright reporter of caspase activity used in E.coli, mammalian cells, and whole organisms which can be used to monitor apoptosis. This reporter, caspase activatable green fluorescent protein (CA-GFP) consists of GFP fused through a flexible linker containing the caspase-3 and -7 recognition sequence, DEVD, to a hydrophobic peptide derived from the influenza A viral M2 protein. This fusion reporter shows a significant fluorescent response in the presence of active caspase. CA-GFP is unique in its ability to hold GFP in a dark state prior to cleavage by active protease. We investigate the mechanism of quenching, examining the structural characteristics which lead to the inability of the GFP chromophore to mature in the presence of the peptide. In better understanding the mechanism of quenching we can engineer CA-GFP to ultimately be used in transgenic animal models. This requires the development of a palette of protease-activatable fluorescent proteins (PrA-FP) which would enable the monitoring of multiple proteolytic events within a cell or organism in real time. Our development of this palette of reporters, varying in their fluorescence and proteolytic response shows that CA-GFP has the potential to be a powerful tool for the study of the role of apoptosis during development in whole organism models and could be an important tool in understanding the role of individual proteases within the complex biochemical environment in the cell.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:open_access_dissertations-1729 |
| Date | 01 February 2013 |
| Creators | Nicholls, Samantha Elizabeth Bernard |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Open Access Dissertations |
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