Center of Innovative Research, Banyan Biomarkers Inc, 12085 Research Drive, Alachua, FL 32615, USA.
Apoptosis. 2010 Dec;15(12):1480-93. doi: 10.1007/s10495-010-0526-4.
Neuronal cell death after traumatic brain injury, Alzheimer's disease and ischemic stroke may in part be mediated through endoplasmic reticulum (ER) stress and unfolded protein response (UPR). UPR results in induction of molecular chaperone GRP78 and the ER-resident caspase-12, whose activation has been proposed to be mediated by calpain and caspase processing, although their relative contribution remains unclear. In this study we induced ER stress with thapsigargin (TG), and determined the activation profile of calpain-2, caspase-3, caspase-7, and caspase-12 by analyses of protein levels, corresponding substrates and breakdown products (BDP). Specific calpain and caspase activity was assessed by analysis of αII-spectrin BDP of 145 kDa (SBDP145), BDP of 150 kDa (SBDP150) and BDP of 120 kDa (SBDP120). Decrease in pro-calpain-2 protein and increased SBDP145 levels by 3 h after TG treatment indicated early calpain activity. Active caspase-7 (p20) increase occurred after 8 h, followed by concomitant up-regulation of active caspase-3 and SBDP120 after 24 h. In vitro digestion experiments supported that SBDP120 was exclusively generated by active caspase-3 and validated that kinectin and co-chaperone p23 were calpain and caspase-7 substrates, respectively. Pro-caspase-12 protein processing by the specific action of calpain and caspase-3/7 was observed in a time-dependent manner. N-terminal pro-domain processing of pro-caspase-12 by calpain generated a 38 kDa fragment, while caspase-3/7 generated a 35 kDa fragment. Antibody developed specifically against the caspase-3/7 C-terminal cleavage site D(341) detected the presence of large subunit (p20) containing 23 kDa fragment that increased after 24 h of TG treatment. Significant caspase-12 enzyme activity was only detected after 24 h of TG treatment and was completely inhibited by caspase 3/7 inhibitor DEVD-fmk and partially by calpain inhibitor SNJ-1945. ER-stress-induced cell death pathway in TG-treated PC12 cells was characterized by up-regulation of GRP-78 and processing and activation of caspase-12 by the orchestrated proteolytic activity of calpain-2 and caspase-3/7.
创伤性脑损伤、阿尔茨海默病和缺血性中风后神经元细胞死亡可能部分是通过内质网（ER）应激和未折叠蛋白反应（UPR）介导的。UPR 导致分子伴侣 GRP78 和 ER 驻留半胱天冬酶-12 的诱导，其激活被提议通过钙蛋白酶和半胱天冬酶处理介导，尽管它们的相对贡献仍不清楚。在这项研究中，我们用他普西加汀（TG）诱导 ER 应激，并通过分析蛋白水平、相应的底物和断裂产物（BDP）来确定钙蛋白酶-2、半胱天冬酶-3、半胱天冬酶-7 和半胱天冬酶-12 的激活情况。通过分析 145 kDa 的 αII- spectrin BDP（SBDP145）、150 kDa 的 BDP（SBDP150）和 120 kDa 的 BDP（SBDP120）来评估特定的钙蛋白酶和半胱天冬酶活性。TG 处理后 3 小时，原钙蛋白酶-2 蛋白减少，SBDP145 水平增加，表明早期钙蛋白酶活性。8 小时后出现活性半胱天冬酶-7（p20）增加，随后 24 小时同时出现活性半胱天冬酶-3 和 SBDP120 的上调。体外消化实验支持 SBDP120 仅由活性半胱天冬酶-3 产生，并验证了 kinectin 和共伴侣 p23 分别是钙蛋白酶和半胱天冬酶-7 的底物。原半胱天冬酶-12 的蛋白加工通过钙蛋白酶和半胱天冬酶-3/7 的特异性作用以时间依赖性方式进行。钙蛋白酶对原半胱天冬酶-12 的 N 端前肽进行特异性处理，生成 38 kDa 片段，而半胱天冬酶-3/7 生成 35 kDa 片段。针对半胱天冬酶-3/7 C 端切割位点 D（341）特异性开发的抗体检测到存在含有 23 kDa 片段的大亚基（p20），该片段在 TG 处理 24 小时后增加。仅在 TG 处理 24 小时后才检测到显著的半胱天冬酶-12 酶活性，并且被半胱天冬酶-3/7 抑制剂 DEVD-fmk 完全抑制，部分被钙蛋白酶抑制剂 SNJ-1945 抑制。用 TG 处理 PC12 细胞的 ER 应激诱导的细胞死亡途径的特征是 GRP-78 的上调以及钙蛋白酶-2 和半胱天冬酶-3/7 的协调蛋白水解活性对半胱天冬酶-12 的加工和激活。
