The major calpain isozymes are long-lived proteins. Design of an antisense strategy for calpain depletion in cultured cells.
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.
Zhang
W
W
Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43699-0008, USA.
Lane
R D
RD
Mellgren
R L
RL
D000595:Amino Acid Sequence; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002154:Calpain / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D003978:Diazomethane / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D005347:Fibroblasts / Q000201:enzymology; D006207:Half-Life; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology; D011487:Protein Conformation
D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; D007527:Isoenzymes; D016376:Oligonucleotides, Antisense; D009842:Oligopeptides; C090218:benzyloxycarbonylleucyl-leucyl-tyrosine diazomethyl ketone; D003978:Diazomethane; C033668:calpastatin; D002154:Calpain