Skip to main content
SpringerLink
Log in

In Vitro Characterization of Chain Depolymerization Activities of SUMO-Specific Proteases

  • Protocol
  • First Online:
SUMO

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1475))

Abstract

SUMO-specific proteases, known as Ulps in baker’s yeast and SENPs in humans, have important roles in controlling the dynamics of SUMO-modified proteins. They display distinct modes of action and specificity, in that they may act on the SUMO precursor, mono-sumoylated, and/or polysumoylated proteins, and they might be specific for substrates with certain SUMO paralogs. SUMO chains may be dismantled either by endo or exo mechanisms. Biochemical characterization of a protease usually requires purification of the protein of interest. Developing a purification protocol, however, can be very difficult, and in some cases, isolation of a protease in its pure form may go along with a substantial loss of activity. To characterize the reaction mechanism of Ulps, we have developed an in vitro assay, which makes use of substrates endowed with artificial poly-SUMO chains of defined lengths, and S. cerevisiae Ulp enzymes in crude extract from E. coli. This fast and economic approach should be applicable to SUMO-specific proteases from other species as well.

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 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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. Eifler K, Vertegaal AC (2015) Mapping the SUMOylated landscape. FEBS J 282(19):3669–3680. doi:10.1111/febs.13378

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Hilgarth RS, Murphy LA, Skaggs HS et al (2004) Regulation and function of SUMO modification. J Biol Chem 279(52):53899–53902. doi:10.1074/jbc.R400021200

    Article  CAS  PubMed  Google Scholar 

  3. Flotho A, Melchior F (2013) Sumoylation: a regulatory protein modification in health and disease. Annu Rev Biochem 82:357–385. doi:10.1146/annurev-biochem-061909-093311

    Article  CAS  PubMed  Google Scholar 

  4. Vertegaal AC (2010) SUMO chains: polymeric signals. Biochem Soc Trans 38(Pt 1):46–49. doi:10.1042/BST0380046

    Article  CAS  PubMed  Google Scholar 

  5. Praefcke GJ, Hofmann K, Dohmen RJ (2012) SUMO playing tag with ubiquitin. Trends Biochem Sci 37(1):23–31. doi:10.1016/j.tibs.2011.09.002

    Article  CAS  PubMed  Google Scholar 

  6. Cheng CH, Lo YH, Liang SS et al (2006) SUMO modifications control assembly of synaptonemal complex and polycomplex in meiosis of Saccharomyces cerevisiae. Genes Dev 20(15):2067–2081. doi:10.1101/gad.1430406

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Klug H, Xaver M, Chaugule VK et al (2013) Ubc9 sumoylation controls SUMO chain formation and meiotic synapsis in Saccharomyces cerevisiae. Mol Cell 50(5):625–636. doi:10.1016/j.molcel.2013.03.027

    Article  CAS  PubMed  Google Scholar 

  8. Bylebyl GR, Belichenko I, Johnson ES (2003) The SUMO isopeptidase Ulp2 prevents accumulation of SUMO chains in yeast. J Biol Chem 278(45):44113–44120. doi:10.1074/jbc.M308357200

    Article  CAS  PubMed  Google Scholar 

  9. Uzunova K, Göttsche K, Miteva M et al (2007) Ubiquitin-dependent proteolytic control of SUMO conjugates. J Biol Chem 282(47):34167–34175. doi:10.1074/jbc.M706505200

    Article  CAS  PubMed  Google Scholar 

  10. Perry JJ, Tainer JA, Boddy MN (2008) A SIM-ultaneous role for SUMO and ubiquitin. Trends Biochem Sci 33(5):201–208. doi:10.1016/j.tibs.2008.02.001

    Article  CAS  PubMed  Google Scholar 

  11. Hunter T, Sun H (2008) Crosstalk between the SUMO and ubiquitin pathways. Ernst Schering Found Symp Proc 1:1–16

    Article  Google Scholar 

  12. Geoffroy MC, Hay RT (2009) An additional role for SUMO in ubiquitin-mediated proteolysis. Nat Rev Mol Cell Biol 10(8):564–568. doi:10.1038/nrm2707

    Article  CAS  PubMed  Google Scholar 

  13. Sriramachandran AM, Dohmen RJ (2014) SUMO-targeted ubiquitin ligases. Biochim Biophys Acta 1843(1):75–85. doi:10.1016/j.bbamcr.2013.08.022

    Article  CAS  PubMed  Google Scholar 

  14. Johnson ES, Schwienhorst I, Dohmen RJ, Blobel G (1997) The ubiquitin-like protein Smt3p is activated for conjugation to other proteins by an Aos1p/Uba2p heterodimer. EMBO J 16(18):5509–5519. doi:10.1093/emboj/16.18.5509

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Shin EJ, Shin HM, Nam E et al (2012) DeSUMOylating isopeptidase: a second class of SUMO protease. EMBO Rep 13(4):339–346. doi:10.1038/embor.2012.3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Li SJ, Hochstrasser M (1999) A new protease required for cell-cycle progression in yeast. Nature 398(6724):246–251. doi:10.1038/18457

    Article  CAS  PubMed  Google Scholar 

  17. Li SJ, Hochstrasser M (2000) The yeast ULP2 (SMT4) gene encodes a novel protease specific for the ubiquitin-like Smt3 protein. Mol Cell Biol 20(7):2367–2377

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Schwienhorst I, Johnson ES, Dohmen RJ (2000) SUMO conjugation and deconjugation. Mol Gen Genet 263(5):771–786

    Article  CAS  PubMed  Google Scholar 

  19. Mukhopadhyay D, Dasso M (2007) Modification in reverse: the SUMO proteases. Trends Biochem Sci 32(6):286–295. doi:10.1016/j.tibs.2007.05.002

    Article  CAS  PubMed  Google Scholar 

  20. Hay RT (2007) SUMO-specific proteases: a twist in the tail. Trends Cell Biol 17(8):370–376. doi:10.1016/j.tcb.2007.08.002

    Article  CAS  PubMed  Google Scholar 

  21. Yeh ET (2009) SUMOylation and De-SUMOylation: wrestling with life’s processes. J Biol Chem 284(13):8223–8227. doi:10.1074/jbc.R800050200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kim JH, Baek SH (2009) Emerging roles of desumoylating enzymes. Biochim Biophys Acta 1792(3):155–162. doi:10.1016/j.bbadis.2008.12.008

    Article  CAS  PubMed  Google Scholar 

  23. Kolli N, Mikolajczyk J, Drag M et al (2010) Distribution and paralogue specificity of mammalian deSUMOylating enzymes. Biochem J 430(2):335–344. doi:10.1042/BJ20100504

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Nayak A, Muller S (2014) SUMO-specific proteases/isopeptidases: SENPs and beyond. Genome Biol 15(7):422. doi:10.1186/s13059-014-0422-2

    Article  PubMed  PubMed Central  Google Scholar 

  25. Saitoh H, Hinchey J (2000) Functional heterogeneity of small ubiquitin-related protein modifiers SUMO-1 versus SUMO-2/3. J Biol Chem 275(9):6252–6258

    Article  CAS  PubMed  Google Scholar 

  26. Tatham MH, Jaffray E, Vaughan OA et al (2001) Polymeric chains of SUMO-2 and SUMO-3 are conjugated to protein substrates by SAE1/SAE2 and Ubc9. J Biol Chem 276(38):35368–35374. doi:10.1074/jbc.M104214200

    Article  CAS  PubMed  Google Scholar 

  27. Seifert A, Schofield P, Barton GJ, Hay RT (2015) Proteotoxic stress reprograms the chromatin landscape of SUMO modification. Sci Signal 8(384):rs7. doi:10.1126/scisignal.aaa2213

    Article  PubMed  Google Scholar 

  28. Eckhoff J, Dohmen RJ (2015) In vitro studies reveal a sequential mode of chain processing by the yeast SUMO (Small Ubiquitin-related Modifier)-specific protease Ulp2. J Biol Chem 290(19):12268–12281. doi:10.1074/jbc.M114.622217

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Wohlschlegel JA, Johnson ES, Reed SI, Yates JR III (2006) Improved identification of SUMO attachment sites using C-terminal SUMO mutants and tailored protease digestion strategies. J Proteome Res 5(4):761–770. doi:10.1021/pr050451o

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Stepanenko OV, Kuznetsova IM, Verkhusha VV, Turoverov KK (2013) Beta-barrel scaffold of fluorescent proteins: folding, stability and role in chromophore formation. Int Rev Cell Mol Biol 302:221–278. doi:10.1016/B978-0-12-407699-0.00004-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgement

J. E. was supported by a predoctoral fellowship from the NRW graduate school IGS DHD.

Author information

Authors and Affiliations

  1. Institute for Genetics, Biocenter, University of Cologne, 50674, Cologne, Germany

    Julia Eckhoff & R. Jürgen Dohmen

Authors
  1. Julia Eckhoff
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. R. Jürgen Dohmen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to R. Jürgen Dohmen .

Editor information

Editors and Affiliations

  1. INBIOMED, San Sebastian, Spain

    Manuel S. Rodriguez

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Eckhoff, J., Dohmen, R.J. (2016). In Vitro Characterization of Chain Depolymerization Activities of SUMO-Specific Proteases. In: Rodriguez, M. (eds) SUMO. Methods in Molecular Biology, vol 1475. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6358-4_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6358-4_9

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6356-0

  • Online ISBN: 978-1-4939-6358-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation