Skip to main content
SpringerLink
Log in

Scalable In Vitro Proteasome Activity Assay

  • Protocol
  • First Online:
The Ubiquitin Proteasome System

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1844))

Abstract

We developed a degradation assay based on fluorescent protein substrates that are efficiently recognized, unfolded, translocated, and hydrolyzed by the proteasome. The substrates consist of three components: a proteasome-binding tag, a folded domain, and an initiation region. All the components of the model substrate can be changed to modulate degradation, and the assay can be performed in parallel in 384-well plates. These properties allow the assay to be used to explore a wide range of experimental conditions and to screen proteasome modulators.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Baumeister W, Walz J, Zuhl F et al (1998) The proteasome: paradigm of a self-compartmentalizing protease. Cell 92:367–380. https://doi.org/10.1016/S0092-8674(00)80929-0

    Article  CAS  PubMed  Google Scholar 

  2. Finley D, Chen X, Walters KJ (2016) Gates, channels, and switches: elements of the proteasome machine. Trends Biochem Sci 41:77–93. https://doi.org/10.1016/j.tibs.2015.10.009

    Article  CAS  PubMed  Google Scholar 

  3. Beck F, Unverdorben P, Bohn S et al (2012) Near-atomic resolution structural model of the yeast 26S proteasome. Proc Natl Acad Sci U S A 109:14870–14875. https://doi.org/10.1073/pnas.1213333109

    Article  PubMed  PubMed Central  Google Scholar 

  4. Lander GC, Estrin E, Matyskiela ME et al (2012) Complete subunit architecture of the proteasome regulatory particle. Nature 482:186–191. https://doi.org/10.1038/nature10774

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Groll M, Bajorek M, Köhler A et al (2000) A gated channel into the proteasome core particle. Nat Struct Mol Biol 7:1062–1067. https://doi.org/10.1038/80992

    Article  CAS  Google Scholar 

  6. Groll M, Ditzel L, Löwe J et al (1997) Structure of 20S proteasome from yeast at 2.4 A resolution. Nature 386:463–471. https://doi.org/10.1038/386463a0

    Article  CAS  PubMed  Google Scholar 

  7. Bhattacharyya S, Yu H, Mim C, Matouschek A (2014) Regulated protein turnover: snapshots of the proteasome in action. Nat Rev Mol Cell Biol 15:122–133. https://doi.org/10.1038/nrm3741

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Hershko A, Ciechanover A (2003) The ubiquitin system. Annu Rev Biochem 67:425–479. https://doi.org/10.1146/annurev.biochem.67.1.425

    Article  Google Scholar 

  9. DeMartino GN, Gillette TG (2007) Proteasomes: machines for all reasons. Cell 129:659–662. https://doi.org/10.1016/j.cell.2007.05.007

    Article  CAS  PubMed  Google Scholar 

  10. Glickman MH, Ciechanover A (2002) The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev 82:373–428. https://doi.org/10.1152/physrev.00027.2001

    Article  CAS  Google Scholar 

  11. Wolf DH, Hilt W (2004) The proteasome: a proteolytic nanomachine of cell regulation and waste disposal. Biochim Biophys Acta 1695:19–31. https://doi.org/10.1016/j.bbamcr.2004.10.007

    Article  CAS  PubMed  Google Scholar 

  12. Suraweera A, Münch C, Hanssum A, Bertolotti A (2012) Failure of amino acid homeostasis causes cell death following proteasome inhibition. Mol Cell 48(2):242–253. https://doi.org/10.1016/j.molcel.2012.08.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Goldberg AL, Rock KL (1992) Proteolysis, proteasomes and antigen presentation. Nature 357:375–379. https://doi.org/10.1038/357375a0

    Article  CAS  PubMed  Google Scholar 

  14. Schmidt M, Finley D (2014) Regulation of proteasome activity in health and disease. Biochim Biophys Acta 1843:13–25. https://doi.org/10.1016/j.bbamcr.2013.08.012

    Article  CAS  PubMed  Google Scholar 

  15. Dantuma NP, Bott LC (2014) The ubiquitin-proteasome system in neurodegenerative diseases: precipitating factor, yet part of the solution. Front Mol Neurosci 7:1345. https://doi.org/10.3389/fnmol.2014.00070

    Article  CAS  Google Scholar 

  16. Powell SR, Herrmann J, Lerman A et al (2012) The ubiquitin–proteasome system and cardiovascular disease. In: The proteasomal system in aging and disease. Elsevier, Amsterdam, pp 295–346

    Chapter  Google Scholar 

  17. Cromm PM, Crews CM (2017) The proteasome in modern drug discovery: second life of a highly valuable drug target. ACS Cent Sci 3:830–838. https://doi.org/10.1021/acscentsci.7b00252

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Shah JJ, Orlowski RZ (2009) Proteasome inhibitors in the treatment of multiple myeloma. Leukemia 23:1964–1979. https://doi.org/10.1038/leu.2009.173

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Liggett A, Crawford LJ, walker B et al (2010) Methods for measuring proteasome activity: current limitations and future developments. Leuk Res 34:1403–1409. https://doi.org/10.1016/j.leukres.2010.07.003

    Article  CAS  PubMed  Google Scholar 

  20. Kisselev AF, Goldberg AL (2005) Monitoring activity and inhibition of 26S proteasomes with fluorogenic peptide substrates. Meth Enzymol 398:364–378. https://doi.org/10.1016/S0076-6879(05)98030-0

    Article  CAS  PubMed  Google Scholar 

  21. Saeki Y, Isono E, Toh-E A (2005) Preparation of ubiquitinated substrates by the PY motif-insertion method for monitoring 26S proteasome activity. Meth Enzymol 399:215–227. https://doi.org/10.1016/S0076-6879(05)99014-9

    Article  CAS  PubMed  Google Scholar 

  22. Martinez-Fonts K, Matouschek A (2016) A rapid and versatile method for generating proteins with defined ubiquitin chains. Biochemistry 55:1898–1908. https://doi.org/10.1021/acs.biochem.5b01310

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Prakash S, Tian L, Ratliff KS et al (2004) An unstructured initiation site is required for efficient proteasome-mediated degradation. Nat Struct Mol Biol 11:830–837. https://doi.org/10.1038/nsmb814

    Article  CAS  PubMed  Google Scholar 

  24. Singh Gautam AK, Balakrishnan S, Venkatraman P (2012) Direct ubiquitin independent recognition and degradation of a folded protein by the eukaryotic proteasomes-origin of intrinsic degradation signals. PLoS One 7:e34864–e34814. https://doi.org/10.1371/journal.pone.0034864

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Li Y, Tomko RJ, Hochstrasser M (2015) Proteasomes: isolation and activity assays. Curr Protoc Cell Biol 4:3.43.1–3.43.20. https://doi.org/10.1002/0471143030.cb0343s67

    Article  Google Scholar 

  26. Verma R, McDonald H, Yates JR, Deshaies RJ (2001) Selective degradation of ubiquitinated Sic1 by purified 26S proteasome yields active S phase cyclin-Cdk. Mol Cell 8:439–448

    Article  CAS  Google Scholar 

  27. Hanna J, Hathaway NA, Tone Y et al (2006) Deubiquitinating enzyme Ubp6 functions noncatalytically to delay proteasomal degradation. Cell 127:99–111. https://doi.org/10.1016/j.cell.2006.07.038

    Article  CAS  PubMed  Google Scholar 

  28. Kraut DA, Israeli E, Schrader EK et al (2012) Sequence- and species-dependence of proteasomal processivity. ACS Chem Biol 7:1444–1453. https://doi.org/10.1021/cb3001155

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Tian L, Holmgren RA, Matouschek A (2005) A conserved processing mechanism regulates the activity of transcription factors Cubitus interruptus and NF-κB. Nat Struct Mol Biol 12:1045–1053. https://doi.org/10.1038/nsmb1018

    Article  CAS  PubMed  Google Scholar 

  30. Schrader EK, Harstad KG, Holmgren RA, Matouschek A (2011) A three-part signal governs differential processing of Gli1 and Gli3 proteins by the proteasome. J Biol Chem 286:39051–39058. https://doi.org/10.1074/jbc.M111.274993

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Yu H, Singh Gautam AK, Wilmington SR et al (2016) Conserved sequence preferences contribute to substrate recognition by the proteasome. J Biol Chem 291:14526–14539. https://doi.org/10.1074/jbc.M116.727578

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Wilmington SR, Matouschek A (2016) An inducible system for rapid degradation of specific cellular proteins using proteasome adaptors. PLoS One 11:e0152679–e0152616. https://doi.org/10.1371/journal.pone.0152679

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Yu H, Kago G, Yellman CM, Matouschek A (2016) Ubiquitin-like domains can target to the proteasome but proteolysis requires a disordered region. EMBO J 35:1522–1536. https://doi.org/10.15252/embj.201593147

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Beckwith R, Estrin E, Worden EJ, Martin A (2013) Reconstitution of the 26S proteasome reveals functional asymmetries in its AAA+ unfoldase. Nat Publ Group 20:1164–1172. https://doi.org/10.1038/nsmb.2659

    Article  CAS  Google Scholar 

  35. Neefjes J, Dantuma NP (2004) Fluorescent probes for proteolysis: tools for drug discovery. Nat Rev Drug Discov 3:58–69. https://doi.org/10.1038/nrd1282

    Article  CAS  PubMed  Google Scholar 

  36. Bhattacharyya S, Renn JP, Yu H et al (2016) An assay for 26S proteasome activity based on fluorescence anisotropy measurements of dye-labeled protein substrates. Anal Biochem 509:50–59. https://doi.org/10.1016/j.ab.2016.05.026

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Stack JH, Whitney M, Rodems SM, Pollok BA (2000) A ubiquitin-based tagging system for controlled modulation of protein stability. Nat Biotechnol 18:1298–1302. https://doi.org/10.1038/82422

    Article  CAS  PubMed  Google Scholar 

  38. Fishbain S, Prakash S, Herrig A et al (2011) Rad23 escapes degradation because it lacks a proteasome initiation region. Nat Commun 2:192–199. https://doi.org/10.1038/ncomms1194

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Sone T, Saeki Y, Toh-e A, Yokosawa H (2004) Sem1p is a novel subunit of the 26 S proteasome from Saccharomyces cerevisiae. J Biol Chem 279:28807–28816. https://doi.org/10.1074/jbc.M403165200

    Article  CAS  PubMed  Google Scholar 

  40. Inobe T, Fishbain S, Prakash S, Matouschek A (2011) Defining the geometry of the two-component proteasome degron. Nat Chem Biol 7:161–167. https://doi.org/10.1038/nchembio.521

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Thrower JS, Hoffman L, Rechsteiner M, Pickart CM (2000) Recognition of the polyubiquitin proteolytic signal. EMBO J 19:94–102. https://doi.org/10.1093/emboj/19.1.94

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Nager AR, Baker TA, Sauer RT (2011) Stepwise unfolding of a β barrel protein by the AAA+ ClpXP protease. J Mol Biol 413:4–16. https://doi.org/10.1016/j.jmb.2011.07.041

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Fishbain S, Inobe T, Israeli E et al (2015) Sequence composition of disordered regions fine-tunes protein half-life. Nat Struct Mol Biol 22:214–221. https://doi.org/10.1038/nsmb.2958

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Fischer M, Hilt W, Richter-Ruoff B et al (1994) The 26S proteasome of the yeast Saccharomyces cerevisiae. FEBS Lett 355:69–75. https://doi.org/10.1016/0014-5793(94)01177-X

    Article  CAS  PubMed  Google Scholar 

  45. Ostuka Y, Homma N, Shiga K et al (1998) Purification and properties of rabbit muscle proteasome, and its effect on myofibrillar structure. Meat Sci 49:365–378. https://doi.org/10.1016/S0309-1740(97)00141-1

    Article  Google Scholar 

  46. Yang P (2003) Purification of the Arabidopsis 26 S proteasome: biochemical and molecular analyses revealed the presence of multiple isoforms. J Biol Chem 279:6401–6413. https://doi.org/10.1074/jbc.M311977200

    Article  CAS  PubMed  Google Scholar 

  47. DeMartino GN, Proske RJ, Moomaw CR et al (1996) Identification, purification, and characterization of a PA700-dependent activator of the proteasome. J Biol Chem 271:3112–3118. https://doi.org/10.1074/jbc.271.6.3112

    Article  CAS  PubMed  Google Scholar 

  48. Hirano Y, Murata S, Tanaka K (2005) Large- and small-scale purification of mammalian 26S proteasomes. Meth Enzymol 399:227–240. https://doi.org/10.1016/S0076-6879(05)99015-0

    Article  CAS  PubMed  Google Scholar 

  49. Besche HC, Goldberg AL (2012) Affinity purification of mammalian 26S proteasomes using an ubiquitin-like domain. In: cDNA Libraries. Humana Press, Totowa, NJ, pp 423–432

    Google Scholar 

  50. Besche HC, Haas W, Gygi SP, Goldberg AL (2009) Isolation of mammalian 26S proteasomes and p97/VCP complexes using the ubiquitin-like domain from HHR23B reveals novel proteasome-associated proteins. Biochemistry 48:2538–2549. https://doi.org/10.1021/bi802198q

    Article  CAS  PubMed  Google Scholar 

  51. Leggett DS, Hanna J, Borodovsky A et al (2002) Multiple associated proteins regulate proteasome structure and function. Mol Cell 10:495–507

    Article  CAS  Google Scholar 

  52. Leggett DS, Glickman MH, Finley D (2005) Purification of proteasomes, proteasome subcomplexes, and proteasome-associated proteins from budding yeast. Methods Mol Biol 301:57–70. https://doi.org/10.1385/1-59259-895-1:057

    Article  CAS  PubMed  Google Scholar 

  53. Verma R, Chen S, Feldman R et al (2000) Proteasomal proteomics: identification of nucleotide-sensitive proteasome-interacting proteins by mass spectrometric analysis of affinity-purified proteasomes. Mol Biol Cell 11:3425–3439

    Article  CAS  Google Scholar 

  54. Elsasser S, Schmidt M, Finley D (2005) Characterization of the proteasome using native gel electrophoresis. Meth Enzymol 398:353–363. https://doi.org/10.1016/S0076-6879(05)98029-4

    Article  CAS  PubMed  Google Scholar 

  55. 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. https://doi.org/10.1074/jbc.M112.441907

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Benaroudj N, Zwickl P, Seemuller E et al (2003) ATP hydrolysis by the proteasome regulatory complex PAN serves multiple functions in protein degradation. Mol Cell 11:69–78. https://doi.org/10.1016/S1097-2765(02)00775-X

    Article  CAS  PubMed  Google Scholar 

  57. Takahashi K, Matouschek A, Inobe T (2015) Regulation of proteasomal degradation by modulating proteasomal initiation regions. ACS Chem Biol 10:2537–2543. https://doi.org/10.1021/acschembio.5b00554

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Johnson KA (2009) Fitting enzyme kinetic data with KinTek Global Kinetic Explorer. Meth Enzymol 467:601–626. https://doi.org/10.1016/S0076-6879(09)67023-3

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgment

This work was supported by U54 GM105816, R21 CA191664, R21 CA196456, and R01 GM124501 from the National Institutes of Health; RP140328 from the Cancer Prevention and Research Institute of Texas (CPRIT); and F-1817 from the Welch.

Author information

Authors and Affiliations

  1. Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA

    Amit Kumar Singh Gautam, Kirby Martinez-Fonts & Andreas Matouschek

Authors
  1. Amit Kumar Singh Gautam
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kirby Martinez-Fonts
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Andreas Matouschek
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Andreas Matouschek .

Editor information

Editors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada

    Thibault Mayor

  2. Department of Chemistry and Biochemistry, University of Nevada Las Vegas, Las Vegas, NV, USA

    Gary Kleiger

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Singh Gautam, A.K., Martinez-Fonts, K., Matouschek, A. (2018). Scalable In Vitro Proteasome Activity Assay. 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_21

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8706-1_21

  • 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

Publish with us

Policies and ethics

Search

Navigation