The major calpain isozymes are long-lived proteins - Design of an antisense strategy for calpain depletion in cultured cells
The major calpain isozymes are long-lived proteins - Design of an antisense strategy for calpain depletion in cultured cells
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DOI：
10.1074/jbc.271.31.18825
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发表时间：
1996-08-02
影响因子：
4.8
通讯作者：
Mellgren, RL
中科院分区：
文献类型：
--
作者：
Zhang, WL;Lane, RD;Mellgren, RL
钙蛋白酶是细胞内钙依赖性蛋白酶，被认为参与钙相关的信号转导途径。已经提出钙蛋白酶是通过自蛋白水解机制激活的。如果这是真的，那么人们将预期钙蛋白酶蛋白在细胞中的半衰期相对较短。为了验证这一假设，用[S-35]甲硫氨酸脉冲标记WI-38人二倍体成纤维细胞，结果表明，钙蛋白酶的两种主要同工酶m-钙蛋白酶和mu-钙蛋白酶的代谢半衰期约为5天，钙蛋白酶在几种人细胞系A-431、HeLa、VA-13、C-33 A、此外，钙蛋白酶抑制蛋白calpastatin也具有较长的代谢半衰期，提示钙蛋白酶通过自身蛋白水解激活的模型需要重新研究。基于对钙蛋白酶代谢稳定性的了解，设计了一种将WI-38细胞和HeLa细胞长期暴露于钙蛋白酶小亚基反义寡脱氧核苷酸的方案。钙蛋白酶小亚基在处理5天或更长时间后的消耗导致细胞增殖的抑制，这可以通过从培养基中去除反义寡脱氧核糖核苷酸来逆转。与先前的研究一起，这些结果表明哺乳动物细胞增殖需要钙蛋白酶。
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.