Cross-talk between calpain and caspase proteolytic systems during neuronal apoptosis
Cross-talk between calpain and caspase proteolytic systems during neuronal apoptosis
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DOI：
10.1074/jbc.m212255200
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发表时间：
2003-04-18
影响因子：
4.8
通讯作者：
Siman, R
中科院分区：
文献类型：
--
作者：
Neumar, RW;Xu, YA;Siman, R
钙蛋白酶和半胱天冬酶蛋白水解系统之间的串扰使得确定它们在细胞凋亡中的不同作用的工作变得复杂。这项研究检查了过表达钙蛋白酶抑制剂（特定的内源性钙蛋白酶抑制剂）对细胞凋亡刺激后两个蛋白水解系统活性的影响。用全长人钙蛋白酶 eDNA 稳定转染人 SH-SY5Y 神经母细胞瘤细胞，根据蛋白质印迹结果，细胞提取物中的钙蛋白酶抑制活性高出 20 倍，且钙蛋白酶抑制活性高出 5 倍。野生型和钙蛋白酶抑制素过表达 (CST1) 细胞进行神经元分化，并用十字孢菌素 (0.1-1.0 muM) 诱导细胞凋亡。 Calpastatin 过度表达降低了 calpain 激活，增加了 caspase-3 样活性，并加速了细胞凋亡核形态的出现。与分化的野生型细胞相比，0.1-0.2 muM 星形孢菌素后，分化的 CST1 中基于钙黄绿素-乙酰氧基甲基荧光的质膜完整性在 24 小时时显着更高。然而，当星形孢菌素剂量较高（0.5-1.0 μM）时，这种保护作用就会消失，导致过表达的卡蛋白酶介导的半胱天冬酶介导的明显降解。这些结果表明钙蛋白酶在神经元凋亡过程中具有双重作用。在早期执行阶段，钙蛋白酶下调 caspase-3 样活性并减缓细胞凋亡核形态的进展。随后的钙蛋白酶活性，由半胱天冬酶介导的钙蛋白酶抑制素降解促进，导致质膜破坏和继发性坏死。
Cross-talk between calpain and caspase proteolytic systems has complicated efforts to determine their distinct roles in apoptotic cell death. This study examined the effect of overexpressing calpastatin, the specific endogenous calpain inhibitor, on the activity of the two proteolytic systems following an apoptotic stimulus. Human SH-SY5Y neuroblastoma cells were stably transfected with full-length human calpastatin eDNA resulting in 20-fold overexpression based on Western blot and 5-fold greater calpain inhibitory activity in cell extracts. Wild type and calpastatin overexpressing (CST1) cells were neuronally differentiated and apoptosis-induced with staurosporine (0.1-1.0 muM). Calpastatin overexpression decreased calpain activation, increased caspase-3-like activity, and accelerated the appearance of apoptotic nuclear morphology. Following 0.1-0.2 muM staurosporine, plasma membrane integrity based on calcein-acetoxymethyl fluorescence was significantly greater at 24 h in differentiated CST1 compared with differentiated wild type cells. However, this protective effect was lost at higher staurosporine doses (0.5-1.0 muM), which resulted in pronounced caspase-mediated degradation of the overexpressed calpastatin. These results suggest a dual role for calpains during neuronal apoptosis. In the early execution phase, calpain down-regulates caspase-3-like activity and slows progression of apoptotic nuclear morphology. Subsequent calpain activity, facilitated by caspase-mediated degradation of calpastatin, contributes to plasma membrane disruption and secondary necrosis.