Gel-Based protease proteomics for identifying the novel calpain substrates in dopaminergic neuronal cell

Chiho Kim, Nuri Yun, Young Mook Lee, Jae Y. Jeong, Jeong Y. Baek, Hwa Young Song, Chung Ju, Moussa B.H. Youdim, Byung K. Jin, Won Ki Kim, Young J. Oh

Research output: Contribution to journalArticlepeer-review

16 Citations (Scopus)


Calpains are a family of calcium-dependent cysteine proteases that are ubiquitously expressed in mammals and play critical roles in neuronal death by catalyzing substrate proteolysis. Here, we developed two-dimensional gel electrophoresis-based protease proteomics to identify putative calpain substrates. To accomplish this, cellular lysates from neuronal cells were first separated by pI, and the immobilized sample on a gel strip was incubated with a recombinant calpain and separated by molecular weight. Among 25 altered protein spots that were differentially expressed by at least 2-fold, we confirmed that arsenical pump-driving ATPase, optineurin, and peripherin were cleaved by calpain using in vitro and in vivo cleavage assays. Furthermore, we found that all of these substrates were cleaved in MN9D cells treated with either ionomycin or 1-methyl-4-phenylpyridinium, both of which cause a calcium-mediated calpain activation. Their cleavage was blocked by calcium chelator or calpain inhibitors. In addition, calpain-mediated cleavage of these substrates and its inhibition by calpeptin were confirmed in a middle cerebral artery occlusion model of cerebral ischemia, as well as a stereotaxic brain injection model of Parkinson disease. Transient overexpression of each protein was shown to attenuate 1-methyl-4-phenylpyridinium-induced cell death, indicating that these substrates may confer protection of varying magnitudes against dopaminergic injury. Taken together, the data indicate that our protease proteomic method has the potential to be applicable for identifying proteolytic substrates affected by diverse proteases. Moreover, the results described here will help us decipher the molecular mechanisms underlying the progression of neurodegenerative disorders where protease activation is critically involved.

Original languageEnglish
Pages (from-to)36717-36732
Number of pages16
JournalJournal of Biological Chemistry
Issue number51
Publication statusPublished - 2013 Dec 20

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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