Abstract
Because proteasomes catalyze most of the protein degradation in mammalian cells, and their functioning is essential for cellular homeostasis, proteasome structure, biochemical mechanisms, and regulation in normal and disease states are now widely studied and are of major importance. In addition, inhibitors of the proteasome’s peptidase activity have proven to be very valuable as research tools and in the treatment of hematologic malignancies, and a number of newer pharmacological agents that alter proteasome function are being developed. The rapid degradation of ubiquitinated proteins by the 26S proteasome involves multiple enzymatic and non-enzymatic steps, including the binding of ubiquitinated substrates to the 19S particle (Subheading 3.2), opening the gated substrate entry channel into the 20S particle (Subheading 3.3), disassembly of the Ub chain (Subheading 3.4), ATP hydrolysis (Subheading 3.5), substrate unfolding and translocation, and proteolysis within the 20S particle (Subheadings 3.3 and 3.7). Assaying each of these processes is important if we are to fully understand the physiological regulation of proteasome function and the effects of disease or drugs. Here, we describe several methods that we have found useful to measure many of these individual activities using purified proteasomes. Studies using these approaches have already provided valuable new insights into the effects of post-synthetic modifications to 26S subunits, the physiological regulation of the ubiquitin-proteasome system, and the impairment of proteasome activity in neurodegenerative disease. These advances would not have been possible if only the standard assays of peptidase activity were used.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Finley D (2009) Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu Rev Biochem 78:477–513
Smith DM, Benaroudj N, Goldberg A (2006) Proteasomes and their associated ATPases: a destructive combination. J Struct Biol 156:72–83
Kisselev AF, Garcia-Calvo M, Overkleeft HS, Peterson E, Pennington MW, Ploegh HL, Thornberry NA, Goldberg AL (2003) The caspase-like sites of proteasomes, their substrate specificity, new inhibitors and substrates, and allosteric interactions with the trypsin-like sites. J Biol Chem 278:35869–35877
Groll M, Huber R (2003) Substrate access and processing by the 20S proteasome core particle. Int J Biochem Cell Biol 35:606–616
Groll M, Bajorek M, Köhler A, Moroder L, Rubin DM, Huber R, Glickman MH, Finley D (2000) A gated channel into the proteasome core particle. Nat Struct Biol 7:1062–1067
Smith DM, Fraga H, Reis C, Kafri G, Goldberg AL (2011) ATP binds to proteasomal ATPases in pairs with distinct functional effects, implying an ordered reaction cycle. Cell 144:526–538
Matyskiela ME, Lander GC, Martin A (2013) Conformational switching of the 26S proteasome enables substrate degradation. Nat Struct Mol Biol 20:781–788
Peth A, Besche HC, Goldberg AL (2009) Ubiquitinated proteins activate the proteasome by binding to Usp14/Ubp6, which causes 20S gate opening. Mol Cell 36:794–804
Cui Z, Scruggs SB, Gilda JE, Ping P, Gomes AV (2014) Regulation of cardiac proteasomes by ubiquitination, SUMOylation, and beyond. J Mol Cell Cardiol 71:32–42
Collins GA, Goldberg AL (2017) The logic of the 26S proteasome. Cell 169:792–806
Li X, Thompson D, Kumar B, DeMartino GN (2014) Molecular and cellular roles of PI31 (PSMF1) protein in regulation of proteasome function. J Biol Chem 289:17392–17405
Ortega J, Heymann JB, Kajava AV, Ustrell V, Rechsteiner M, Steven AC (2005) The axial channel of the 20S proteasome opens upon binding of the PA200 activator. J Mol Biol 346:1221–1227
Ma CP, Slaughter CA, DeMartino GN (1992) Identification, purification, and characterization of a protein activator (PA28) of the 20 S proteasome (macropain). J Biol Chem 267:10515–10523
Lokireddy S, Kukushkin NV, Goldberg AL (2015) cAMP-induced phosphorylation of 26S proteasomes on Rpn6/PSMD11 enhances their activity and the degradation of misfolded proteins. Proc Natl Acad Sci U S A 112:E7176–E7185
Guo X, Wang X, Wang Z, Banerjee S, Yang J, Huang L, Dixon JE (2016) Site-specific proteasome phosphorylation controls cell proliferation and tumorigenesis. Nat Cell Biol 18:202–212
Besche HC, Sha Z, Kukushkin NV, Peth A, Hock EM, Kim W, Gygi S, Gutierrez JA, Liao H, Dick L, Goldberg AL (2014) Autoubiquitination of the 26S proteasome on Rpn13 regulates breakdown of ubiquitin conjugates. EMBO J 33:1159–1176
Lee C, Schwartz MP, Prakash S, Iwakura M, Matouschek A (2001) ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal. Mol Cell 7:627–637
Peth A, Uchiki T, Goldberg AL (2010) ATP-dependent steps in the binding of ubiquitin conjugates to the 26S proteasome that commit to degradation. Mol Cell 40:671–681
Nathan JA, Kim HT, Ting L, Gygi SP, Goldberg AL (2013) Why do cellular proteins linked to K63-polyubiquitin chains not associate with proteasomes? EMBO J 32:552–565
Deveraux Q, Ustrell V, Pickart C, Rechsteiner M (1994) A 26 S protease subunit that binds ubiquitin conjugates. J Biol Chem 269:7059–7061
Qiu XB, Ouyang SY, Li CJ, Miao S, Wang L, Goldberg AL (2006) hRpn13/ADRM1/GP110 is a novel proteasome subunit that binds the deubiquitinating enzyme, UCH37. EMBO J 25:5742–5753
Shi Y, Chen X, Elsasser S, Stocks BB, Tian G, Lee BH, Shi Y, Zhang N, de Poot SA, Tuebing F, Sun S, Vannoy J, Tarasov SG, Engen JR, Finley D, Walters KJ (2016) Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome. Science 351(6275):aad9421
Erales J, Hoyt MA, Troll F, Coffino P (2012) Functional asymmetries of proteasome translocase pore. J Biol Chem 287:18535–18543
Kisselev AF, Goldberg AL (2001) Proteasome inhibitors: from research tools to drug candidates. Chem Biol 8:739–758
Kisselev AF, Goldberg AL (2005) Monitoring activity and inhibition of 26S proteasomes with fluorogenic peptide substrates. Methods Enzymol 398:364–378
Sledz P, Unverdorben P, Beck F, Pfeifer G, Schweitzer A, Forster F, Baumeister W (2013) Structure of the 26S proteasome with ATP-gammaS bound provides insights into the mechanism of nucleotide-dependent substrate translocation. Proc Natl Acad Sci U S A 110:7264–7269
Smith DM, Chang SC, Park S, Finley D, Cheng Y, Goldberg AL (2007) Docking of the proteasomal ATPases’ carboxyl termini in the 20S proteasome's alpha ring opens the gate for substrate entry. Mol Cell 27:731–744
Kisselev AF, Callard A, Goldberg AL (2006) Importance of the different proteolytic sites of the proteasome and the efficacy of inhibitors varies with the protein substrate. J Biol Chem 281:8582–8590
Borodovsky A, Kessler BM, Casagrande R, Overkleeft HS, Wilkinson KD, Ploegh HL (2001) A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14. EMBO J 20:5187–5196
Li T, Naqvi NI, Yang H, Teo TS (2000) Identification of a 26S proteasome-associated UCH in fission yeast. Biochem Biophys Res Commun 272:270–275
Stone M, Hartmann-Petersen R, Seeger M, Bech-Otschir D, Wallace M, Gordon C (2004) Uch2/Uch37 is the major deubiquitinating enzyme associated with the 26S proteasome in fission yeast. J Mol Biol 344:697–706
Yao T, Cohen RE (2002) A cryptic protease couples deubiquitination and degradation by the proteasome. Nature 419:403–407
Verma R, Aravind L, Oania R, McDonald WH, Yates JR 3rd, Koonin EV, Deshaies RJ (2002) Role of Rpn11 metalloprotease in deubiquitination and degradation by the 26S proteasome. Science 298:611–615
Kuo CL, Goldberg AL (2017) Ubiquitinated proteins promote the association of proteasomes with the deubiquitinating enzyme Usp14 and the ubiquitin ligase Ube3c. Proc Natl Acad Sci U S A 114:E3404–E3413
Lee BH, Lee MJ, Park S, Oh DC, Elsasser S, Chen PC, Gartner C, Dimova N, Hanna J, Gygi SP, Wilson SM, King RW, Finley D (2010) Enhancement of proteasome activity by a small-molecule inhibitor of USP14. Nature 467:179–184
Lee BH, Lu Y, Prado MA, Shi Y, Tian G, Sun S, Elsasser S, Gygi SP, King RW, Finley D (2016) USP14 deubiquitinates proteasome-bound substrates that are ubiquitinated at multiple sites. Nature 532:398–401
Peth A, Kukushkin N, Bossé M, Goldberg AL (2013) Ubiquitinated proteins activate the proteasomal ATPases by binding to Usp14 or Uch37 homologs. J Biol Chem 288:7781–7790
Kim HT, Goldberg AL (2017) The deubiquitinating enzyme Usp14 allosterically inhibits multiple proteasomal activities and ubiquitin-independent proteolysis. J Biol Chem 292:9830–9839
VerPlank JJS, Lokireddy S, Feltri ML, Goldberg AL, Wrabetz L (2018) Impairment of protein degradation and proteasome function in hereditary neuropathies. Glia 66:379–395
Li J, Yakushi T, Parlati F, Mackinnon AL, Perez C, Ma Y, Carter KP, Colayco S, Magnuson G, Brown B, Nguyen K, Vasile S, Suyama E, Smith LH, Sergienko E, Pinkerton AB, Chung TDY, Palmer AE, Pass I, Hess S, Cohen SM, Deshaies RJ (2017) Capzimin is a potent and specific inhibitor of proteasome isopeptidase Rpn11. Nat Chem Biol 13:486–493
Perez C, Li J, Parlati F, Rouffet M, Ma Y, Mackinnon AL, Chou TF, Deshaies RJ, Cohen SM (2017) Discovery of an inhibitor of the proteasome subunit Rpn11. J Med Chem 60:1343–1361
Benaroudj N, Zwickl P, Seemuller E, Baumeister W, Goldberg AL (2003) ATP hydrolysis by the proteasome regulatory complex PAN serves multiple functions in protein degradation. Mol Cell 11:69–78
Goldberg AL (1992) The mechanism and functions of ATP-dependent proteases in bacterial and animal cells. Eur J Biochem 203:9–23
Menon AS, Goldberg AL (1987) Protein substrates activate the ATP-dependent protease La by promoting nucleotide binding and release of bound ADP. J Biol Chem 262:14929–14934
Peth A, Nathan JA, Goldberg AL (2013) The ATP costs and time required to degrade ubiquitinated proteins by the 26 S proteasome. J Biol Chem 288:29215–29222
Lanzetta PA, Alvarez LJ, Reinach PS, Candia OA (1979) An improved assay for nanomole amounts of inorganic phosphate. Anal Biochem 100:95–97
Saeki Y, Isono E, Toh EA (2005) Preparation of ubiquitinated substrates by the PY motif-insertion method for monitoring 26S proteasome activity. Methods Enzymol 399:215–227
Bhattacharyya S, Renn JP, Yu H, Marko JF, Matouschek A (2016) An assay for 26S proteasome activity based on fluorescence anisotropy measurements of dye-labeled protein substrates. Anal Biochem 509:50–59
Chung CH, Goldberg AL (1981) The product of the lon (capR) gene in Escherichia coli is the ATP-dependent protease, protease La. Proc Natl Acad Sci U S A 78:4931–4935
Saeki Y, Tayama Y, Toh-e A, Yokosawa H (2004) Definitive evidence for Ufd2-catalyzed elongation of the ubiquitin chain through Lys48 linkage. Biochem Biophys Res Commun 320:840–845
Acknowledgments
The methods described here were developed through research support to our laboratory from the NIH-NIGMS (R01 GM51923), Cure Alzheimer's Fund, Muscular Dystrophy Association (MDA-419143), and Project A.L.S (2015-06).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Kim, H.T., Collins, G.A., Goldberg, A.L. (2018). Measurement of the Multiple Activities of 26S Proteasomes. In: Mayor, T., Kleiger, G. (eds) The Ubiquitin Proteasome System. Methods in Molecular Biology, vol 1844. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8706-1_19
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8706-1_19
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8705-4
Online ISBN: 978-1-4939-8706-1
eBook Packages: Springer Protocols