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

Abstract

Calpain-1 and -2 are Ca-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.

GNAO1organizes the cytoskeletal remodeling and firing of developing neurons

“…To this end, we analyzed the time course for the cleavage pattern of Sptan1 after treating the cells with 5 µM ionomycin, a potent activator of calcium ionophore and calpains. 39,40 We confirmed that 15 to 30 minutes of ionomycin treatments induced the cleavage of Sptan1 in the control cells, whereas such responses disappeared in Gnao1-deficient cells ( Figure 2D,E).…”

Developing fluorescence sensor probe to capture activated muscle-specific calpain-3 (CAPN3) in living muscle cells

“…The Förster/fluorescence resonance energy transfer (FRET) technique has been applied for the detection of protease activity of conventional CAPNs, CAPN1 and CAPN2, to demonstrate real time proteolysis in vitro and in vivo (Bartoli et al, 2006;McCartney and Davies, 2019;McCartney et al, 2018a;Vanderklish et al, 2000). As for detection of activated CAPN3, our group has previously generated a prototype FRET-based sensor probe (SP) that is cleaved in in vitro test tubes by CAPN3 and CAPN3 splice variant lacking exons 6, 15 and 16 (Ono et al, 2004).…”

Calcium-triggered DNA-mediated membrane fusion in synthetic cells

“…The ability to cleave CCS was validated first by conjugating it to a fluorescence resonance energy transfer (FRET) dye pair (DABCYL and EDANS). [13][14][15] When mixed with calpain-1 and CaCl 2 in a bulk reaction, the CCS was cleaved, leading to a 2.5-fold increase in EDANS fluorescence over the control condition (Fig. S4A, ESI †).…”