Abstract
Calpains are intracellular Ca2+-dependent proteases that are thought to participate in Ca2+-associated signal transduction pathways. It has been proposed that calpains are activated by an autoproteolytic mechanism. If this is true one would expect a relatively short half-life for calpain protein in cells. To test this hypothesis, WI-38 human diploid fibroblasts were pulse-labeled with [35S]methionine, and calpain was immunoprecipitated at various times after chasing with nonradioactive methionine to determine residual radioactivity. The results demonstrated that the two major calpain isozymes, m-calpain and micro-calpain, had metabolic half-lives of approximately 5 days. Calpains were long-lived proteins in several human cell lines, A-431, HeLa, VA-13, C-33A, and TE2 cells. In addition, calpastatin, the calpain-specific inhibitor protein, also had a long metabolic half-life. These observations suggest that the model for calpain activation by autoproteolysis requires re-investigation. Based on a knowledge of calpain metabolic stability, a protocol was devised for chronic exposure of WI-38 cells and HeLa cells to a calpain small subunit antisense oligodeoxyribonucleotide. Depletion of calpain small subunit after 5 or more days of treatment led to inhibition of cell proliferation that could be reversed by removal of antisense oligodeoxyribonucleotide from the culture medium. Together with previous studies, these results indicate a requirement for calpains in mammalian cell proliferation.
Highlights
Calpains are intracellular Ca2؉-dependent proteases that are thought to participate in Ca2؉-associated signal transduction pathways
WI-38 human diploid fibroblasts were pulse-labeled with [35S]methionine, and calpain was immunoprecipitated at various times after chasing with nonradioactive methionine to determine residual radioactivity
Based on a knowledge of calpain metabolic stability, a protocol was devised for chronic exposure of WI-38 cells and HeLa cells to a calpain small subunit antisense oligodeoxyribonucleotide
Summary
From the Departments of Pharmacology and Therapeutics and ‡Anatomy, Medical College of Ohio, Toledo, Ohio 43699-0008. It has been proposed that calpains are activated by an autoproteolytic mechanism If this is true one would expect a relatively short half-life for calpain protein in cells. Calpastatin, the calpain-specific inhibitor protein, had a long metabolic half-life These observations suggest that the model for calpain activation by autoproteolysis requires re-investigation. If calpains are reasonably active within cells, the autolysis model suggests that they should have relatively short metabolic half-lives To address this issue, and as a preliminary study to establish conditions for calpain depletion by ASODN1 methodology, we have investigated the metabolic stability of calpains in WI-38 fibroblasts and several human cell lines. In addition to showing that calpains are relatively stable proteins, these studies have allowed the development of a protocol for depletion of calpain in cultured cells by chronic exposure to a calpain AS-ODN
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