Calpain-truncated CRMP-3 and-4 contribute to potassium deprivation-induced apoptosis of cerebellar granule neurons
Calpain-truncated CRMP-3 and-4 contribute to potassium deprivation-induced apoptosis of cerebellar granule neurons
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DOI：
10.1002/pmic.200800979
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发表时间：
2009-07-01
期刊：
影响因子：
3.4
通讯作者：
Li, Mingtao
中科院分区：
文献类型：
--
作者：
Liu, Wei;Zhou, Xing-wang;Li, Mingtao
越来越多的证据表明，钙蛋白酶介导的选择性蛋白质的蛋白水解加工在神经元凋亡中起着重要作用。研究钙蛋白酶介导的切割事件及其相关功能有助于更好地理解神经元凋亡和神经退行性疾病。因此，我们研究了钙蛋白酶底物在钾剥夺诱导的小脑颗粒神经元（CGNs）凋亡中的作用。通过2-D DIGE和MALDI-TOF/TOF MS分析鉴定了12种已知的钙蛋白酶底物和7种新的钙蛋白酶底物。此外，通过Western印迹分析验证了所鉴定的新型钙蛋白酶底物。此外，我们专注于在体内和体外的钙蛋白酶，但不是由半胱天冬酶的蛋白质水解CRMPs反应介质蛋白（CRMP-1，-2，-3和-4亚型）。为了阐明钙蛋白酶介导的CRMPs蛋白水解的性质，我们构建了CRMPs的缺失突变体用于进一步的生化研究。体外切割试验表明，CRMP-1，-2和-4被钙蛋白酶在C-末端截短，而CRMP-3在N-末端被切割。最后，我们通过在CGN中过表达截短的CRMPs来评估CRMPs在钾剥夺触发的神经元凋亡过程中的作用。我们的数据清楚地表明，截短的CRMP-3和-4，而不是CRMP-1和-2，显着诱导神经细胞凋亡。这些发现表明，当CGN发生凋亡时，钙蛋白酶截短的CRMP-3和CRMP-4作为促凋亡参与者。
Increasing evidence shows that calpain-mediated proteolytic processing of a selective number of proteins plays an important role in neuronal apoptosis. Study of calpain-mediated cleavage events and related functions may contribute to a better understanding of neuronal apoptosis and neurodegenerative diseases. We, therefore, investigated the role of calpain substrates in potassium deprivation-induced apoptosis of cerebellar granule neurons (CGNs). Twelve previously known and seven novel candidates of calpain substrates were identified by 2-D DIGE and MALDI-TOF/TOF MS analysis. Further, the identified novel calpain substrates were validated by Western blot analysis. Moreover, we focused on the collapsin response mediator proteins (CRMP-1, -2, -3 and -4 isoforms) and found that CRMPs were proteolytically processed by calpain but not by caspase, both in vivo and in vitro. To clarify the properties of the calpain-mediated proteolysis of CRMPs, we constructed the deletion mutants of CRMPs for additional biochemical studies. In vitro cleavage assays revealed that CRMP-1, -2 and -4 were truncated by calpain at the C-terminus, whereas CRMP-3 was cleaved at the N-terminus. Finally, we assessed the role of CRMPs in the process of potassium deprivation-triggered neuronal apoptosis by overexpressing the truncated CRMPs in CGNs. Our data clearly showed that the truncated CRMP-3 and -4, but not CRMP-1 and -2, significantly induced neuronal apoptosis. These findings demonstrated that calpain-truncated CRMP-3 and -4 act as pro-apoptotic players when CGNs undergo apoptosis.