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An easy-to-use FRET protein substrate to detect calpain cleavage in vitro and in vivo
Authors:Christian-Scott E McCartney  James A MacLeod  Peter A Greer  Peter L Davies
Institution:1. Department of Biomedical and Molecular Sciences, Queen''s University, Kingston, ON K7L 3N6, Canada;2. Department of Pathology and Molecular Medicine, Queen''s University, Kingston, ON K7L 3N6, Canada
Abstract: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.
Keywords:FRET  Fluorescent Resonance Energy Transfer  PLFAAR  optimal calpain cleavage sequence in the one-letter amino acid code  CFP  Cyan Fluorescent Protein  YFP  Yellow Fluorescent Protein  SDS-PAGE  sodium dodecyl sulfate polyacrylamide gel electrophoresis  EDANS  5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid small molecule fluorophore  DABCYL  4-([4-(Dimethylamino)phenyl]azo)benzoic acid succinimidyl ester small molecule quencher of EDANS  EDTA  ethylenediaminetetraacetic acid  PMSF  phenylmethylsulphonyl fluoride  NTA  nitrilotriacetic acid  GeCKO  genome-scale CRISPR knock-out  CAST4  calpastatin fourth inhibitory domain  GAPDH  glyceraldehyde-3-phosphate dehydrogenase  MDA-MB-231  human breast adenocarcinoma cell line  Calpain  Substrate  Cysteine protease  Cleavage specificity  Substrate optimization  Fret
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