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Succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (SLLVY-AMC) is a fluorogenic substrate used to measure calpain activity and the "chymotrypsin-like" activity of the 20s proteasome. The goal of this study was to determine the relative role of calpains and the proteasome on SLLVY-AMC cleavage in attached and suspended renal epithelial cells (NRK-52E). The proteasome inhibitor epoxomicin did not inhibit purified calpain 1 or calpain 10 cleavage of SLLVY-AMC. Epoxomicin inhibited 11% of total SLLVY-AMC cleavage in attached cells and increasing concentrations of the calpain inhibitor calpeptin were additive. In contrast, cell suspensions had a 3.5-fold higher rate of SLLVY-AMC cleavage, epoxomicin inhibited cleavage 65% and calpeptin inhibited cleavage an additional 26%. Calpeptin alone also inhibited proteasomal activity. In conclusion, (1) SLLVY-AMC is cleaved in cells by calpain and the proteasome, (2) proteasome activity can be measured with epoxomicin, and (3) calpeptin can inhibit proteasome activity in some cases; thus limiting the use of SLLVY-AMC and calpeptin.

Calpains are calcium- and thiol-dependent proteases that cleave a variety of intracellular substrates. Overactivation of the calpains has been implicated in a number of diseases and conditions such as ischemic stroke indicating a need for the development of calpain inhibitors. A major problem with current calpain inhibitors has been specific targeting to calpain. To identify highly specific calpain interacting peptides, we developed a peptide-phage library screening method based on the calcium-dependent conformation change associated with calpain activation. A phage-peptide library representing greater than 2 billion expressed 12-mers was incubated with calpain I in the presence of calcium. The calcium-dependent bound phage was then eluted by addition of EGTA. After four rounds of selection we found a conserved 5-mer sequence represented by LSEAL. Synthetic LSEAL inhibited tau-calpain interaction and in vitro proteolysis of tau- and alpha-synuclein by calpains. Deletion of the portion of the tau protein containing a homologous sequence to LSEAL resulted in decreased calpain-mediated tau degradation. These data suggest that these peptides may represent novel calpastatin mimetics.

Calpain was generally believed to exist and function only in the cytoplasm. However, m-calpain has now been detected in the extracellular spaces of some kinds of tissue. In this study, we demonstrated the existence of m-calpain in the medium surrounding MC3T3-E1 cultures, and its activity by zymography. At the same time, the amount of lactate dehydrogenase in medium of MC3T3-E1 culture was extremely low compared with other cell cultures, suggesting that m-calpain found in the culture medium of MC3T3-E1 cells originated mainly from active secretion. Moreover, the secretion of m-calpain was not blocked by brefeldin A, implying that m-calpain may be secreted by a nonclassical pathway. Recently, MC3T3-E1 has been reported to produce matrix vesicles and media vesicles, and we demonstrated m-calpain in these vesicles produced by MC3T3-E1 cultures. We therefore concluded that these vesicles are partly responsible for the secretion of m-calpain into the culture medium of MC3T3-E1 cells., (Copyright 2001 Academic Press.)

We have developed a sensitive and continuously recording fluorogenic assay for the thiol protease calpain. This assay uses the dipeptide substrate Suc-Leu-Tyr-4-Methoxy-2-Naphthylamine (Suc-LY-MNA) in Tris buffer (pH 7.5) in the presence of 0.1% CHAPS. The assay is linear over a wide range of enzyme concentration and is capable of detecting 10 picomolar calpain making it more sensitive than any previously published method. Moreover, this assay gives a rate that is linear for over ten minutes making it useful for mechanistic studies of inhibitors. This assay can be easily adapted to a 96-well plate format facilitating the large scale screening of inhibitors., (Copyright 1998 Academic Press.)