The major calpain isozymes are long-lived proteins - Design of an antisense strategy for calpain depletion in cultured cells

被引:73
作者
Zhang, WL
Lane, RD
Mellgren, RL
机构
[1] MED COLL OHIO, DEPT PHARMACOL, TOLEDO, OH 43690 USA
[2] MED COLL OHIO, DEPT THERAPEUT, TOLEDO, OH 43690 USA
[3] MED COLL OHIO, DEPT ANAT, TOLEDO, OH 43690 USA
关键词
D O I
10.1074/jbc.271.31.18825
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 [S-35]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 mu-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.
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页码:18825 / 18830
页数:6
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