Skip to main content
Top
Published in:

2015 | Online First | Chapter

26S and PA28-20S Proteasome Activity in Cytosolic Extracts from Embryonic Stem Cells

Author : Malin Hernebring

Published in: Methods in Molecular Biology™

Publisher: Springer New York

Activate our intelligent search to find suitable subject content or patents.

search-config
loading …

Abstract

The proteasome is a complex multisubunit protease that plays a major role in the degradation of proteins in eukaryotic cells. Proteasome function is one of the key players regulating the proteome and it is vital for many cellular processes. The method described here makes it possible to assay the proteolytic capacities of proteasome complexes separately in crude cytosolic extracts from ES cells. The method is based on hydrolysis of a fluorogenic peptide substrate in lysates prepared under conditions that favor the interactions of the 20S proteasomal catalytical core with either the 19S or the PA28αβ proteasome regulator.
Literature
1.
3.
go back to reference Pickering AM, Linder RA, Zhang H, Forman HJ, Davies KJ (2012) Nrf2-dependent induction of proteasome and Pa28alphabeta regulator are required for adaptation to oxidative stress. J Biol Chem 287:10021PubMedCentralCrossRefPubMed Pickering AM, Linder RA, Zhang H, Forman HJ, Davies KJ (2012) Nrf2-dependent induction of proteasome and Pa28alphabeta regulator are required for adaptation to oxidative stress. J Biol Chem 287:10021PubMedCentralCrossRefPubMed
4.
go back to reference Li J, Powell SR, Wang X (2011) Enhancement of proteasome function by PA28α overexpression protects against oxidative stress. FASEB J 25:883 Li J, Powell SR, Wang X (2011) Enhancement of proteasome function by PA28α overexpression protects against oxidative stress. FASEB J 25:883
5.
go back to reference Brooks P, Fuertes G, Murray RZ, Bose S, Knecht E, Rechsteiner MC, Hendil KB, Tanaka K, Dyson J, Rivett J (2000) Subcellular localization of proteasomes and their regulatory complexes in mammalian cells. Biochem J 346(1):155PubMedCentralCrossRefPubMed Brooks P, Fuertes G, Murray RZ, Bose S, Knecht E, Rechsteiner MC, Hendil KB, Tanaka K, Dyson J, Rivett J (2000) Subcellular localization of proteasomes and their regulatory complexes in mammalian cells. Biochem J 346(1):155PubMedCentralCrossRefPubMed
6.
go back to reference Chen X, Barton LF, Chi Y, Clurman BE, Roberts JM (2007) Ubiquitin-independent degradation of cell-cycle inhibitors by the REGγ proteasome. Mol Cell 26:843 Chen X, Barton LF, Chi Y, Clurman BE, Roberts JM (2007) Ubiquitin-independent degradation of cell-cycle inhibitors by the REGγ proteasome. Mol Cell 26:843
9.
go back to reference Tanahashi N, Murakami Y, Minami Y, Shimbara N, Hendil KB, Tanaka K (2000) Hybrid proteasomes. Induction by interferon-gamma and contribution to ATP-dependent proteolysis. J Biol Chem 275:14336CrossRefPubMed Tanahashi N, Murakami Y, Minami Y, Shimbara N, Hendil KB, Tanaka K (2000) Hybrid proteasomes. Induction by interferon-gamma and contribution to ATP-dependent proteolysis. J Biol Chem 275:14336CrossRefPubMed
10.
go back to reference Blickwedehl J, Agarwal M, Seong C, Pandita RK, Melendy T, Sung P, Pandita TK, Bangia N (2008) Role for proteasome activator PA200 and postglutamyl proteasome activity in genomic stability. Proc Natl Acad Sci U S A 105:16165PubMedCentralCrossRefPubMed Blickwedehl J, Agarwal M, Seong C, Pandita RK, Melendy T, Sung P, Pandita TK, Bangia N (2008) Role for proteasome activator PA200 and postglutamyl proteasome activity in genomic stability. Proc Natl Acad Sci U S A 105:16165PubMedCentralCrossRefPubMed
11.
go back to reference Bence NF, Sampat RM, Kopito RR (2001) Impairment of the ubiquitin-proteasome system by protein aggregation. Science 292:1552CrossRefPubMed Bence NF, Sampat RM, Kopito RR (2001) Impairment of the ubiquitin-proteasome system by protein aggregation. Science 292:1552CrossRefPubMed
12.
go back to reference Eisele F, Wolf DH (2008) Degradation of misfolded protein in the cytoplasm is mediated by the ubiquitin ligase Ubr1. FEBS Lett 582:4143CrossRefPubMed Eisele F, Wolf DH (2008) Degradation of misfolded protein in the cytoplasm is mediated by the ubiquitin ligase Ubr1. FEBS Lett 582:4143CrossRefPubMed
13.
go back to reference Djakovic SN, Schwarz LA, Barylko B, DeMartino GN, Patrick GN (2009) Regulation of the proteasome by neuronal activity and calcium/calmodulin-dependent protein kinase II. J Biol Chem 284:26655PubMedCentralCrossRefPubMed Djakovic SN, Schwarz LA, Barylko B, DeMartino GN, Patrick GN (2009) Regulation of the proteasome by neuronal activity and calcium/calmodulin-dependent protein kinase II. J Biol Chem 284:26655PubMedCentralCrossRefPubMed
14.
go back to reference Bose S, Brooks P, Mason GG, Rivett AJ (2001) Gamma-Interferon decreases the level of 26 S proteasomes and changes the pattern of phosphorylation. Biochem J 353:291PubMedCentralPubMed Bose S, Brooks P, Mason GG, Rivett AJ (2001) Gamma-Interferon decreases the level of 26 S proteasomes and changes the pattern of phosphorylation. Biochem J 353:291PubMedCentralPubMed
15.
go back to reference Rivett AJ, Bose S, Pemberton AJ, Brooks P, Onion D, Shirley D, Stratford FLL, Forti K (2002) Assays of proteasome activity in relation to aging. Exp Gerontol 37:1217CrossRefPubMed Rivett AJ, Bose S, Pemberton AJ, Brooks P, Onion D, Shirley D, Stratford FLL, Forti K (2002) Assays of proteasome activity in relation to aging. Exp Gerontol 37:1217CrossRefPubMed
16.
go back to reference Rosner M, Hengstschlager M (2008) Cytoplasmic and nuclear distribution of the protein complexes mTORC1 and mTORC2: rapamycin triggers dephosphorylation and delocalization of the mTORC2 components rictor and sin1. Hum Mol Genet 17:2934CrossRefPubMed Rosner M, Hengstschlager M (2008) Cytoplasmic and nuclear distribution of the protein complexes mTORC1 and mTORC2: rapamycin triggers dephosphorylation and delocalization of the mTORC2 components rictor and sin1. Hum Mol Genet 17:2934CrossRefPubMed
17.
go back to reference Lodish H, Berk A, Zipursky SL, Matsudaira P, Baltimore D, Darnell JE (2000) Molecular cell biology. WH Freeman, New York Lodish H, Berk A, Zipursky SL, Matsudaira P, Baltimore D, Darnell JE (2000) Molecular cell biology. WH Freeman, New York
18.
go back to reference Pickering AM, Koop AL, Teoh CY, Ermak G, Grune T, Davies KJA (2010) The immunoproteasome, the 20S proteasome and the PA28αβ proteasome regulator are oxidative-stress-adaptive proteolytic complexes. Biochem J 432:585 Pickering AM, Koop AL, Teoh CY, Ermak G, Grune T, Davies KJA (2010) The immunoproteasome, the 20S proteasome and the PA28αβ proteasome regulator are oxidative-stress-adaptive proteolytic complexes. Biochem J 432:585
19.
go back to reference Kisselev AF, Goldberg AL (2005) Monitoring activity and inhibition of 26S proteasomes with fluorogenic peptide substrates. Methods Enzymol 398:364CrossRefPubMed Kisselev AF, Goldberg AL (2005) Monitoring activity and inhibition of 26S proteasomes with fluorogenic peptide substrates. Methods Enzymol 398:364CrossRefPubMed
20.
go back to reference Hernebring M, Fredriksson Å, Liljevald M, Cvijovic M, Norrman K, Wiseman J, Semb H, Nyström T (2013) Removal of damaged proteins during ES cell fate specification requires the proteasome activator PA28. Sci Rep 3:1381PubMedCentralCrossRefPubMed Hernebring M, Fredriksson Å, Liljevald M, Cvijovic M, Norrman K, Wiseman J, Semb H, Nyström T (2013) Removal of damaged proteins during ES cell fate specification requires the proteasome activator PA28. Sci Rep 3:1381PubMedCentralCrossRefPubMed
21.
go back to reference Reinheckel T, Sitte N, Ullrich O, Kuckelkorn U, Davies KJ, Grune T (1998) Comparative resistance of the 20S and 26S proteasome to oxidative stress. Biochem J 335:637PubMedCentralCrossRefPubMed Reinheckel T, Sitte N, Ullrich O, Kuckelkorn U, Davies KJ, Grune T (1998) Comparative resistance of the 20S and 26S proteasome to oxidative stress. Biochem J 335:637PubMedCentralCrossRefPubMed
22.
go back to reference Ferrington DA, Husom AD, Thompson LV (2005) Altered proteasome structure, function, and oxidation in aged muscle. FASEB J 19:644PubMed Ferrington DA, Husom AD, Thompson LV (2005) Altered proteasome structure, function, and oxidation in aged muscle. FASEB J 19:644PubMed
23.
go back to reference Dick LR, Cruikshank AA, Grenier L, Melandri FD, Nunes SL, Stein RL (1996) Mechanistic studies on the inactivation of the proteasome by lactacystin: a central role for clasto-lactacystin beta-lactone. J Biol Chem 271:7273CrossRefPubMed Dick LR, Cruikshank AA, Grenier L, Melandri FD, Nunes SL, Stein RL (1996) Mechanistic studies on the inactivation of the proteasome by lactacystin: a central role for clasto-lactacystin beta-lactone. J Biol Chem 271:7273CrossRefPubMed
24.
go back to reference Meng L, Mohan R, Kwok BH, Elofsson M, Sin N, Crews CM (1999) Epoxomicin, a potent and selective proteasome inhibitor, exhibits in vivo antiinflammatory activity. Proc Natl Acad Sci U S A 96:10403PubMedCentralCrossRefPubMed Meng L, Mohan R, Kwok BH, Elofsson M, Sin N, Crews CM (1999) Epoxomicin, a potent and selective proteasome inhibitor, exhibits in vivo antiinflammatory activity. Proc Natl Acad Sci U S A 96:10403PubMedCentralCrossRefPubMed
26.
go back to reference Bulteau AL, Lundberg KC, Humphries KM, Sadek HA, Szweda PA, Friguet B, Szweda LI (2001) Oxidative modification and inactivation of the proteasome during coronary occlusion/reperfusion. J Biol Chem 276:30057CrossRefPubMed Bulteau AL, Lundberg KC, Humphries KM, Sadek HA, Szweda PA, Friguet B, Szweda LI (2001) Oxidative modification and inactivation of the proteasome during coronary occlusion/reperfusion. J Biol Chem 276:30057CrossRefPubMed
27.
go back to reference Rodgers KJ, Dean RT (2003) Assessment of proteasome activity in cell lysates and tissue homogenates using peptide substrates. Int J Biochem Cell Biol 35:716CrossRefPubMed Rodgers KJ, Dean RT (2003) Assessment of proteasome activity in cell lysates and tissue homogenates using peptide substrates. Int J Biochem Cell Biol 35:716CrossRefPubMed
Metadata
Title
26S and PA28-20S Proteasome Activity in Cytosolic Extracts from Embryonic Stem Cells
Author
Malin Hernebring
Copyright Year
2015
Publisher
Springer New York
DOI
https://doi.org/10.1007/7651_2015_216