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 journalArticle

11 Citations (Scopus)

Abstract

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
Volume288
Issue number51
DOIs
Publication statusPublished - 2013 Dec 20

Fingerprint

Calpain
Proteomics
Peptide Hydrolases
Gels
Substrates
1-Methyl-4-phenylpyridinium
Peripherins
Chemical activation
Proteolysis
Arsenicals
Calcium
Ionomycin
Mammals
Cysteine Proteases
Middle Cerebral Artery Infarction
Electrophoresis, Gel, Two-Dimensional
Cell death
Brain Ischemia
Electrophoresis
Neurodegenerative Diseases

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Kim, C., Yun, N., Lee, Y. M., Jeong, J. Y., Baek, J. Y., Song, H. Y., ... Oh, Y. J. (2013). Gel-Based protease proteomics for identifying the novel calpain substrates in dopaminergic neuronal cell. Journal of Biological Chemistry, 288(51), 36717-36732. https://doi.org/10.1074/jbc.M113.492876

Gel-Based protease proteomics for identifying the novel calpain substrates in dopaminergic neuronal cell. / Kim, Chiho; Yun, Nuri; Lee, Young Mook; Jeong, Jae Y.; Baek, Jeong Y.; Song, Hwa Young; Ju, Chung; Youdim, Moussa B H; Jin, Byung K.; Kim, Won-Ki; Oh, Young J.

In: Journal of Biological Chemistry, Vol. 288, No. 51, 20.12.2013, p. 36717-36732.

Research output: Contribution to journalArticle

Kim, C, Yun, N, Lee, YM, Jeong, JY, Baek, JY, Song, HY, Ju, C, Youdim, MBH, Jin, BK, Kim, W-K & Oh, YJ 2013, 'Gel-Based protease proteomics for identifying the novel calpain substrates in dopaminergic neuronal cell', Journal of Biological Chemistry, vol. 288, no. 51, pp. 36717-36732. https://doi.org/10.1074/jbc.M113.492876
Kim, Chiho ; Yun, Nuri ; Lee, Young Mook ; Jeong, Jae Y. ; Baek, Jeong Y. ; Song, Hwa Young ; Ju, Chung ; Youdim, Moussa B H ; Jin, Byung K. ; Kim, Won-Ki ; Oh, Young J. / Gel-Based protease proteomics for identifying the novel calpain substrates in dopaminergic neuronal cell. In: Journal of Biological Chemistry. 2013 ; Vol. 288, No. 51. pp. 36717-36732.
@article{7db7ea37350c4caa91c933dfbdfecc58,
title = "Gel-Based protease proteomics for identifying the novel calpain substrates in dopaminergic neuronal cell",
abstract = "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.",
author = "Chiho Kim and Nuri Yun and Lee, {Young Mook} and Jeong, {Jae Y.} and Baek, {Jeong Y.} and Song, {Hwa Young} and Chung Ju and Youdim, {Moussa B H} and Jin, {Byung K.} and Won-Ki Kim and Oh, {Young J.}",
year = "2013",
month = "12",
day = "20",
doi = "10.1074/jbc.M113.492876",
language = "English",
volume = "288",
pages = "36717--36732",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "51",

}

TY - JOUR

T1 - Gel-Based protease proteomics for identifying the novel calpain substrates in dopaminergic neuronal cell

AU - Kim, Chiho

AU - Yun, Nuri

AU - Lee, Young Mook

AU - Jeong, Jae Y.

AU - Baek, Jeong Y.

AU - Song, Hwa Young

AU - Ju, Chung

AU - Youdim, Moussa B H

AU - Jin, Byung K.

AU - Kim, Won-Ki

AU - Oh, Young J.

PY - 2013/12/20

Y1 - 2013/12/20

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84890947271&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84890947271&partnerID=8YFLogxK

U2 - 10.1074/jbc.M113.492876

DO - 10.1074/jbc.M113.492876

M3 - Article

VL - 288

SP - 36717

EP - 36732

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 51

ER -