Skip to main content
SpringerLink
Log in

Determination of Proteasomal Unfolding Ability

  • Protocol
  • First Online:
Targeted Protein Degradation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2365))

Abstract

We use an in vitro degradation assay with a model substrate to assess proteasomal unfolding ability. Our substrate has an unstructured region that is the site of ubiquitination, followed by an easy-to-unfold domain and a difficult-to-unfold domain. Degradation proceeds through the unstructured and easy-to-unfold domains, but the difficult-to-unfold domain can be degraded completely or, if the proteasome stalls, can be released as a partially degraded fragment. The ratio between these two possible outcomes allows us to quantify the unfolding ability and determine how processively the proteasome degrades its substrates.

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 219.99
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. Voges D, Zwickl P, Baumeister W (1999) The 26S proteasome: a molecular machine designed for controlled proteolysis. Annu Rev Biochem 68:1015–1068

    Article  CAS  Google Scholar 

  2. Finley D, Ulrich HD, Sommer T et al (2012) The ubiquitin-proteasome system of Saccharomyces cerevisiae. Genetics 192:319–360

    Article  CAS  Google Scholar 

  3. Finley D (2009) Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu Rev Biochem 78:477–513

    Article  CAS  Google Scholar 

  4. Bard JAM, Goodall EA, Greene ER et al (2018) Structure and function of the 26S proteasome. Annu Rev Biochem 87:697–724

    Article  CAS  Google Scholar 

  5. Lee C, Schwartz MP, Prakash S et al (2001) ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal. Mol Cell 7:627–637

    Article  CAS  Google Scholar 

  6. 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

    Article  CAS  Google Scholar 

  7. Groll M, Bochtler M, Brandstetter H et al (2005) Molecular machines for protein degradation. Chembiochem 6:222–256

    Article  CAS  Google Scholar 

  8. Bar-Nun S, Glickman MH (2012) Proteasomal AAA-ATPases: structure and function. Biochim Biophys Acta 1823(1):67–82

    Article  CAS  Google Scholar 

  9. Matyskiela ME, Martin A (2013) Design principles of a universal protein degradation machine. J Mol Biol 425:199–213

    Article  CAS  Google Scholar 

  10. Finley D, Chen X, Walters KJ (2016) Gates, channels, and switches: elements of the proteasome machine. Trends Biochem Sci 41:77–93

    Article  CAS  Google Scholar 

  11. Wehmer M, Rudack T, Beck F et al (2017) Structural insights into the functional cycle of the ATPase module of the 26S proteasome. Proc Natl Acad Sci U S A 114:1305–1310

    Article  CAS  Google Scholar 

  12. Koodathingal P, Jaffe NE, Kraut DA et al (2009) ATP-dependent proteases differ substantially in their ability to unfold globular proteins. J Biol Chem 284:18674–18684

    Article  CAS  Google Scholar 

  13. Kraut DA, Israeli E, Schrader EK et al (2012) Sequence- and species-dependence of proteasomal processivity. ACS Chem Biol 7:1444–1453

    Article  CAS  Google Scholar 

  14. Reichard EL, Chirico GG, Dewey WJ et al (2016) Substrate ubiquitination controls the unfolding ability of the proteasome. J Biol Chem 291:18547–18561

    Article  CAS  Google Scholar 

  15. Cundiff MD, Hurley CM, Wong JD et al (2019) Ubiquitin receptors are required for substrate-mediated activation of the proteasome's unfolding ability. Sci Rep 9:14506

    Article  Google Scholar 

  16. Vu N-D, Feng H, Bai Y (2004) The folding pathway of Barnase: the rate-limiting transition state and a hidden intermediate under native conditions. Biochemistry 43:3346–3356

    Article  CAS  Google Scholar 

  17. Kim Y, Ho SO, Gassman NR et al (2008) Efficient site-specific labeling of proteins via cysteines. Bioconjug Chem 19:786–791

    Article  CAS  Google Scholar 

  18. Raasi S, Pickart CM (2005) Ubiquitin chain synthesis. Methods Mol Biol 301:47–55

    CAS  PubMed  Google Scholar 

  19. Carvalho AF, Pinto MP, Grou CP et al (2012) High-yield expression in Escherichia coli and purification of mouse ubiquitin-activating enzyme E1. Mol Biotechnol 51:254–261

    Article  CAS  Google Scholar 

  20. Small E, Eggler A, Mesecar AD (2010) Development of an efficient E. coli expression and purification system for a catalytically active, human Cullin3-RINGBox1 protein complex and elucidation of its quaternary structure with Keap1. Biochem Biophys Res Commun 400:471–475

    Article  CAS  Google Scholar 

  21. Eggler AL, Liu G, Pezzuto JM et al (2005) Modifying specific cysteines of the electrophile-sensing human Keap1 protein is insufficient to disrupt binding to the Nrf2 domain Neh2. Proc Natl Acad Sci U S A 102:10070–10075

    Article  CAS  Google Scholar 

  22. Xia Z, Webster A, Du F et al (2008) Substrate-binding sites of UBR1, the ubiquitin ligase of the N-end rule pathway. J Biol Chem 283:24011–24028

    Article  CAS  Google Scholar 

  23. Saeki Y, Isono E, Toh-E A (2005) Preparation of ubiquitinated substrates by the PY motif-insertion method for monitoring 26S proteasome activity. Methods Enzymol 399:215–227

    Article  CAS  Google Scholar 

  24. Kim HC, Huibregtse JM (2009) Polyubiquitination by HECT E3s and the determinants of chain type specificity. Mol Cell Biol 29:3307–3318

    Article  CAS  Google Scholar 

  25. Saeki Y, Kudo T, Sone T et al (2009) Lysine 63-linked polyubiquitin chain may serve as a targeting signal for the 26S proteasome. EMBO J 28:359–371

    Article  CAS  Google Scholar 

  26. Yu H, Matouschek A (2017) Recognition of client proteins by the proteasome. Annu Rev Biophys 46:149–173

    Article  CAS  Google Scholar 

  27. Martinez-Fonts K, Davis C, Tomita T et al (2020) The proteasome 19S cap and its ubiquitin receptors provide a versatile recognition platform for substrates. Nat Commun 11:477

    Article  CAS  Google Scholar 

  28. Zhang DD, Lo S-C, Sun Z et al (2005) Ubiquitination of Keap1, a BTB-Kelch substrate adaptor protein for Cul3, targets Keap1 for degradation by a proteasome-independent pathway. J Biol Chem 280:30091–30099

    Article  CAS  Google Scholar 

  29. Kulathu Y, Komander D (2012) Atypical ubiquitylation - the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages. Nat Rev Mol Cell Biol 13:508–523

    Article  CAS  Google Scholar 

  30. Choi WS, Jeong B-C, Joo YJ et al (2010) Structural basis for the recognition of N-end rule substrates by the UBR box of ubiquitin ligases. Nat Struct Mol Biol 17:1175–1181

    Article  CAS  Google Scholar 

  31. Bodnar NO, Rapoport TA (2017) Molecular mechanism of substrate processing by the Cdc48 ATPase complex. Cell 169:722–735.e9

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors thank Joseph Boscia IV, Mary Cundiff & Eden Reichard for comments and suggestions. This material is based upon work supported by the National Science Foundation under Grants No. 1515229 and 1935596 to DAK.

Author information

Authors and Affiliations

  1. Department of Chemistry, Villanova University, Villanova, PA, USA

    Christina M. Hurley & Daniel A. Kraut

Authors
  1. Christina M. Hurley
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Daniel A. Kraut
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Daniel A. Kraut .

Editor information

Editors and Affiliations

  1. Platform Biology, Arvinas, Inc., New Haven, CT, USA

    Angela M. Cacace

  2. Platform Biology, Arvinas, Inc., New Haven, CT, USA

    Christopher M. Hickey

  3. Platform Biology, Arvinas, Inc., New Haven, CT, USA

    Miklós Békés

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Hurley, C.M., Kraut, D.A. (2021). Determination of Proteasomal Unfolding Ability. In: Cacace, A.M., Hickey, C.M., Békés, M. (eds) Targeted Protein Degradation. Methods in Molecular Biology, vol 2365. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1665-9_12

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1665-9_12

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1664-2

  • Online ISBN: 978-1-0716-1665-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation