An easy-to-use FRET protein substrate to detect calpain cleavage in vitro and in vivo

Publication date: February 2018 Source:Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Volume 1865, Issue 2 Author(s): Christian-Scott E. McCartney, James A. MacLeod, Peter A. Greer, Peter L. Davies Calpain-1 and -2 are Ca2+-activated intracellular cysteine proteases that regulate a wide range of cellular functions through the cleavage of their protein substrates. Unlike degradative proteases, calpains make limited, transformative cleavages, typically in accessible sequences linking discrete subdomains, to irreversibly alter substrate functions. The biological roles of calpain and their interplay with calcium signaling are of significant biomedical interest as biomarkers and potential therapeutic targets in a growing number of diseases including Alzheimer's, cancer and fibrosis. Unfortunately, many of the colorimetric and fluorimetric assays that have been developed to study calpain activity suffer from low sensitivity and/or poor calpain specificity. To address the need for a highly sensitive and calpain-specific substrate suitable for in vitro and in vivo calpain activity analysis, we have developed a protein FRET probe. We inserted the optimized calpain cleavage sequence PLFAAR between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) and modulated its flanking sequences for optimal calpain cleavage. We demonstrate greater sensitivity and calpain-specificity of an optimal 16-residue PLFAAR-based FRET substrate compared to a standard Î...
Source: Biochimica et Biophysica Acta (BBA) Molecular Cell Research - Category: Molecular Biology Source Type: research