Abstract
Proteolysis is a process where proteins are broken down into smaller polypeptides or amino acids, comprising one of the important posttranslational modifications of proteins. Since this process is exquisitely achieved by specialized enzymes called proteases under physiological conditions, abnormal protease activity and dysregulation of their substrate proteins are closely associated with a progression of several neurodegenerative diseases including Alzheimer disease, Parkinson disease, stroke, and spinal cord injury. Thus, it is important to identify the specific substrates of proteases with nonbiased high-throughput screenings to understand how proteolysis contributes to neurodegeneration. Here, we described a so-called gel-based protease proteomic approach. Critical steps of our novel strategy consist of two-dimensional polyacrylamide gel electrophoresis (2-DE)-based protein separation and in vitro incubation with the specific protease of interest. As a prototypic example, cellular lysates obtained from neuronal cells are separated by an isoelectric focusing, and the resulting immobilized proteins on a gel strip are incubated with a predetermined amount of a recombinant or a purified protease. By densitometric analysis of the Coomassie Brilliant Blue-stained gel images following separation by 2-DE, significantly altered protein spots are subjected to a mass spectral analysis for protein identification. Interestingly, the concepts of our strategy can be applied to any proteases, and to any neural cells or neural tissues of one’s interest. Since the immobilized protein spots are exposed to the purified protease, this protocol ensures the identification of only substrates that are directly cleaved by specific protease. This protocol ensures to avoid the possibility of identifying substrates that may be cleaved by combinatorial or sequential activation of proteolytic enzymes present in a liquid state of the lysates. We propose that our strategy can be effectively utilized to provide meaningful insights into newly identified protease substrates and to decipher molecular mechanisms critically involved in neurodegenerative processes.
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References
Shahinian H, Tholen S, Schilling O (2013) Proteomic identification of protease cleavage sites: cell-biological and biomedical applications. Expert Rev Proteomics 10:421–433
Wojcik C, DeMartino GN (2003) Intracellular localization of proteasomes. Int J Biochem Cell Biol 35:579–589
Camins A, Verdaguer E, Folch J, Pallas M (2006) Involvement of calpain activation in neurodegenerative processes. CNS Drug Rev 12:135–148
Puente XS, Sanchez LM, Overall CM, Lopez-Otin C (2003) Human and mouse proteases: a comparative genomic approach. Nat Rev Genet 4:544–558
Samara C, Tavernarakis N (2003) Calcium-dependent and aspartyl proteases in neurodegeneration and ageing in C. elegans. Ageing Res Rev 2:451–471
Wang KK (2000) Calpain and caspase: can you tell the difference? Trends Neurosci 23:20–26
Choi WS, Lee EH, Chung CW, Jung YK, Jin BK, Kim SU, Oh TH, Saido TC, Oh YJ (2001) Cleavage of Bax is mediated by caspase-dependent or -independent calpain activation in dopaminergic neuronal cells: protective role of Bcl-2. J Neurochem 77:1531–1541
Han BS, Hong HS, Choi WS, Markelonis GJ, Oh TH, Oh YJ (2003) Caspase-dependent and -independent cell death pathways in primary cultures of mesencephalic dopaminergic neurons after neurotoxin treatment. J Neurosci 23:5069–5078
Choi WS, Yoon SY, Oh TH, Choi EJ, O'Malley KL, Oh YJ (1999) Two distinct mechanisms are involved in 6-hydroxydopamine- and MPP + −induced dopaminergic neuronal cell death: role of caspases, ROS, and JNK. J Neurosci Res 57:86–94
Han BS, Noh JS, Gwag BJ, Oh YJ (2003) A distinct death mechanism is induced by 1-methyl-4-phenylpyridinium or by 6-hydroxydopamine in cultured rat cortical neurons: degradation and dephosphorylation of tau. Neurosci Lett 341:99–102
Kang H, Han BS, Kim SJ, Oh YJ (2012) Mechanisms to prevent caspase activation in rotenone-induced dopaminergic neurodegeneration: role of ATP depletion and procaspase-9 degradation. Apoptosis 17:449–462
Cryns VL, Byun Y, Rana A, Mellor H, Lustig KD, Ghanem L, Parker PJ, Kirschner MW, Yuan J (1997) Specific proteolysis of the kinase protein kinase C-related kinase 2 by caspase-3 during apoptosis. Identification by a novel, small pool expression cloning strategy. J Biol Chem 272:29449–29453
Kamada S, Kusano H, Fujita H, Ohtsu M, Koya RC, Kuzumaki N, Tsujimoto Y (1998) A cloning method for caspase substrates that uses the yeast two-hybrid system: cloning of the antiapoptotic gene gelsolin. Proc Natl Acad Sci USA 95:8532–8537
Agard NJ, Wells JA (2009) Methods for the proteomic identification of protease substrates. Curr Opin Chem Biol 13:503–509
Mahrus S, Trinidad JC, Barkan DT, Sali A, Burlingame AL, Wells JA (2008) Global sequencing of proteolytic cleavage sites in apoptosis by specific labeling of protein N termini. Cell 134:866–876
Van Damme P, Martens L, Van Damme J, Hugelier K, Staes A, Vandekerckhove J, Gevaert K (2005) Caspase-specific and nonspecific in vivo protein processing during Fas-induced apoptosis. Nat Methods 2:771–777
Celis JE, Ostergaard M, Jensen NA, Gromova I, Rasmussen HH, Gromov P (1998) Human and mouse proteomic databases: novel resources in the protein universe. FEBS Lett 430:64–72
O'Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007–4021
O'Farrell PZ, Goodman HM, O'Farrell PH (1977) High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12:1133–1141
Klose J (1975) Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues A novel approach to testing for induced point mutations in mammals. Humangenetik 26:231–243
Gorg A, Obermaier C, Boguth G, Harder A, Scheibe B, Wildgruber R, Weiss W (2000) The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 21:1037–1053
Klaiman G, Petzke TL, Hammond J, Leblanc AC (2008) Targets of caspase-6 activity in human neurons and Alzheimer disease. Mol Cell Proteomics 7:1541–1555
Lamkanfi M, Kanneganti TD, Van Damme P, Vanden Berghe T, Vanoverberghe I, Vandekerckhove J, Vandenabeele P, Gevaert K, Nunez G (2008) Targeted peptidecentric proteomics reveals caspase-7 as a substrate of the caspase-1 inflammasomes. Mol Cell Proteomics 7:2350–2363
Xu D, Suenaga N, Edelmann MJ, Fridman R, Muschel RJ, Kessler BM (2008) Novel MMP-9 substrates in cancer cells revealed by a label-free quantitative proteomics approach. Mol Cell Proteomics 7:2215–2228
Wood DE, Newcomb EW (1999) Caspase-dependent activation of calpain during drug-induced apoptosis. J Biol Chem 274:8309–8315
Choi HK, Won LA, Kontur PJ, Hammond DN, Fox AP, Wainer BH, Hoffmann PC, Heller A (1991) Immortalization of embryonic mesencephalic dopaminergic neurons by somatic cell fusion. Brain Res 552:67–76
Kim C, Yun N, Lee YM, Jeong JY, Baek JY, Song HY, Ju C, Youdim MB, Jin BK, Kim WK, Oh YJ (2013) Gel-based protease proteomics for identifying the novel calpain substrates in dopaminergic neuronal cell. J Biol Chem 288:36717–36732
Yun N, Lee YM, Kim C, Shibayama H, Tanimura A, Hamanaka Y, Kanakura Y, Park IS, Jo A, Shin JH, Ju C, Kim WK, Oh YJ (2014) Anamorsin, a novel caspase-3 substrate in neurodegeneration. J Biol Chem 289:22183–22195
Chevalier F (2010) Standard dyes for total protein staining in gel-based proteomic analysis. Materials 3:4784–4792
Aebersold R, Mann M (2003) Mass spectrometry-based proteomics. Nature 422:198–207
Acknowledgment
This work was supported by the Ministry for Health, Welfare and Family Affairs (A111382 to YJO) and in part by the Yonsei Intramural Grant (year of 2000 to YJO). We thank Dr. Chung Ju at Korea University for helpful discussion.
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Kim, C., Oh, Y.J. (2017). A Novel 2-DE-Based Proteomic Analysis to Identify Multiple Substrates for Specific Protease in Neuronal Cells. In: Kobeissy, F., Stevens, Jr., S. (eds) Neuroproteomics. Methods in Molecular Biology, vol 1598. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6952-4_10
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DOI: https://doi.org/10.1007/978-1-4939-6952-4_10
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