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Calpain-truncated CRMP-3 and -4 contribute to potassium deprivation-induced apoptosis of cerebellar granule neurons

Authors

  • Wei Liu,

    1. Proteomics Center, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, P. R. China
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    • These authors contributed equally to this work.

  • Xing-wang Zhou,

    1. Proteomics Center, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, P. R. China
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    • These authors contributed equally to this work.

  • Shaojun Liu,

    1. Proteomics Center, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, P. R. China
    2. Department of Pharmacology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, P. R. China
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  • Kunhua Hu,

    1. Proteomics Center, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, P. R. China
    2. Department of Pharmacology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, P. R. China
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  • Chong Wang,

    1. Proteomics Center, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, P. R. China
    2. Department of Pharmacology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, P. R. China
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  • Qingyu He,

    1. Institute of Life and Health Engineering, Jinan University, Guangzhou, P. R. China
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  • Mingtao Li

    Corresponding author
    1. Proteomics Center, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, P. R. China
    2. Department of Pharmacology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, P. R. China
    • Proteomics Center and Department of Pharmacology Zhongshan School of Medicine, Sun Yat-sen University 74 Zhongshan Road II, Guangzhou 510080, P. R. China Fax: +86-20-87331653
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Abstract

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.

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