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 α-
spectrin-based probe. The 16-residue PLFAAR
protein FRET substrate is not significantly cleaved by
trypsin,
chymotrypsin,
cathepsin-L or
caspase-3, and is highly sensitive to both calpain-1 and -2. After transfection of the substrate gene into
breast cancer cells the PLFAAR
protein FRET product was cut in lysed wild-type cells but not in those with a
calpain knock-out phenotype. Blockage of substrate cleavage in the lysates by endogenous and exogenous
calpastatin was observed, and was overcome by adding extra
calpain.