Novel Reagents for the Determination of Calpain Activity In Vitro and In Vivo
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Calpains are multi-domain intracellular Ca2+-activated cysteine proteases. They participate in a number of cellular processes downstream of calcium signaling. Unregulated calpain activity, caused by a loss of calcium homeostasis, has been implicated in a variety of pathologies. For that reason, studying calpain structure-function is of great biomedical interest. Nevertheless, some fundamental questions regarding the behaviors of these proteases remain unanswered largely due to the low sensitivity and poor calpain-specificity of the current methods to measure calpain activity. To address this deficiency, we designed, characterized and optimized a fluorescent protein-based FRET substrate centered on the highly calpain-sensitive PLFAAR cleavage sequence. This reagent proved superior to the endogenous substrate α-spectrin both in vitro and in vivo, making it feasible to accurately determine when, where, and under what circumstances calpain is activated in cells. This substrate may also be useful for the identification of relevant calpain substrates and the assessment of calpain-specific inhibitors in vivo. Calpain-3 is a tissue-enriched isoform whose loss of function causes limb-girdle muscular dystrophy. Due to its instability, largely conferred by its unique insertion sequences, recombinant expression of the full-length enzyme has not been possible. To study the function of insertion sequence one (IS1), we designed and characterized a recombinant calpain-2 surrogate with IS1 inserted into the protease core in its calpain-3-equivalent position. As it does in calpain-3, IS1 occupied the catalytic cleft of the surrogate, restricting access of substrates and inhibitors, establishing its propeptide potential. Surprisingly, the natural calpain inhibitor calpastatin was able to displace IS1 from the active-site and block protease activity. However, this is unlikely to occur in vivo because calpain-3 homodimerizes and presents only one set of PEF domains to hold calpastatin via its anchoring subdomains. We also characterized a protease core from human calpain-3 missing IS1, which showed that IS1 autoinhibition accounts for a nearly 2-fold loss in enzyme activity. Despite the sequence and structural similarities of this protease core with that of calpain-1, small-molecule calpain inhibitors were less effective on this isoform, suggesting that subtle differences in the subsites of the calpain-3 catalytic cleft may confer sufficient isoform-specificity for the development of relatively calpain-3-specific inhibitors.
URI for this recordhttp://hdl.handle.net/1974/24993
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