Abstract
Calpastatin is the natural specific inhibitor of calpain. Recent research has linked uncontrolled calpain activation to tissue damage after neuronal and cardiac ischemias, traumatic spine and brain injuries, as well as Alzheimer's disease and cataract formation. An imbalance between the activities of calpain and calpastatin is believed to be responsible for the pathological role of calpain. An important key to understanding calpain regulation by calpastatin is to determine, at the molecular level, how calpastatin interacts with calpain to inhibit its enzymatic activity. A 27-residue peptide (DPMSSTYIEELGKREVTIPPKYRELLA) derived from subdomain 1B of the repetitive domains of calpain, named peptide B27-WT, was previously shown to be a potent inhibitor of mu- and m-calpain. In this report, a combination of beta-alanine scanning mutagenesis and kinetic measurements was used to probe, in a quantitative, systematic, and simultaneous fashion, the relative contribution of the amino acid side chain and backbone functionalities to the overall calpain-inhibitory activity of B27-WT. The study identified two "hot spots," Leu(11)-Gly(12) and Thr(17)-Ile(18)-Pro(19), in B27-WT within which the residues critical for inhibitory function are clustered. Mutation of any one of the key residues in either of the two hot spots resulted in a dramatic loss of inhibitory activity. Furthermore, it was shown that a restricted conformation of the Leu(11)-Gly(12) and Thr(17)-Ile(18)-Pro(19) backbones is required for the peptide inhibitory function. These results suggest a plausible model in which the two hot spots are situated at or near the interface(s) of the calpain-calpastatin complex and act in a concerted fashion to inhibit calpain. The information on the specific contribution of the amide bond and side chain of each key residue to the bioactivity of B27-WT will contribute to a better understanding of the mechanism of calpain inhibition and lead to novel and effective therapies based on the specific inhibition of dysregulated or overactivated calpain.
Highlights
Calpastatin is the natural specific inhibitor of calpain
The classical - and m-calpains exist as heterodimers consisting of a large (80-kDa) catalytic subunit and a small (28-kDa) regulatory subunit that dissociates in the presence of calcium [7,8,9]
The synthetic peptides were purified to near homogeneity by HPLC, and their molecular weights were confirmed by electrospray ionization and/or matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (Table I)
Summary
Calpastatin is the natural specific inhibitor of calpain. Recent research has linked uncontrolled calpain activation to tissue damage after neuronal and cardiac ischemias, traumatic spine and brain injuries, as well as Alzheimer’s disease and cataract formation. It was shown that a restricted conformation of the Leu11-Gly and Thr17-Ile18-Pro backbones is required for the peptide inhibitory function. These results suggest a plausible model in which the two hot spots are situated at or near the interface(s) of the calpain-calpastatin complex and act in a concerted fashion to inhibit calpain. It has recently been reported that calpastatin resides predominantly in phosphorylated-aggregated bodies, with the inhibitor protein being dephosphorylated and distributed in a Ca2ϩ-dependent manner [11, 12]. Previous studies using bacterially expressed calpastatin fragments have established that the inhibitory activity of calpastatin resides in subdomain B of the repeating structures (domains 1– 4) (15, 20 –23). It has been proposed that these -turns could play an important role in mediating the biological activity of the peptide
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