Calpain and caspase processing of caspase-12 contribute to the ER stress-induced cell death pathway in differentiated PC12 cells
Calpain and caspase processing of caspase-12 contribute to the ER stress-induced cell death pathway in differentiated PC12 cells
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DOI：
10.1007/s10495-010-0526-4
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发表时间：
2010-12-01
期刊：
影响因子：
7.2
通讯作者：
Larner, Stephen F.
中科院分区：
文献类型：
--
作者：
Martinez, Juan A.;Zhang, Zhiqun;Larner, Stephen F.
外伤性脑损伤、阿尔茨海默病和缺血性中风后的神经元细胞死亡可能部分通过内质网（ER）应激和未折叠蛋白反应（UPR）介导。UPR导致分子伴侣GRP78和er驻留caspase-12的诱导，其激活被认为是由calpain和caspase加工介导的，尽管它们的相对贡献尚不清楚。在这项研究中，我们用TG诱导内质网应激，并通过分析蛋白水平、相应底物和分解产物（BDP）来确定calpain-2、caspase-3、caspase-7和caspase-12的激活谱。通过α II-spectrin 145 kDa （SBDP145）、150 kDa （SBDP150）和120 kDa （SBDP120）的BDP分析来评估calpain和caspase的特异性活性。TG处理3小时后，原calpain-2蛋白降低，SBDP145水平升高，表明早期calpain活性。8 h后活性caspase-7 （p20）升高，24 h后活性caspase-3和SBDP120同时上调。体外消化实验证实SBDP120仅由活性caspase-3产生，并验证了激活蛋白和辅伴蛋白p23分别是calpain和caspase-7底物。通过calpain和caspase-3/7的特异性作用，观察到前caspase-12蛋白的加工具有时间依赖性。calpain对pro-caspase-12的n端前结构域加工产生38 kDa的片段，而caspase-3/7产生35 kDa的片段。针对caspase-3/7 c端切割位点D（341）开发的抗体检测到含有23 kDa片段的大亚基（p20），该片段在TG处理24小时后增加。caspase-12酶活性仅在TG处理24 h后检测到，并且被caspase 3/7抑制剂DEVD-fmk完全抑制，部分被calpain抑制剂SNJ-1945抑制。在tg处理的PC12细胞中，内质酰胺应激诱导的细胞死亡通路的特征是GRP-78上调，以及calpain-2和caspase-3/7协同蛋白水解活性加工和激活caspase-12。
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 alpha II-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.