Skip to main content
SpringerLink
Log in

Profiling the Activity of Deubiquitinating Enzymes Using Chemically Synthesized Ubiquitin-Based Probes

  • Protocol
  • First Online:
Activity-Based Proteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1491))

Abstract

Deubiquitinating enzymes (DUBs) are of interest as potential new targets for pharmacological intervention. Active-site-directed probes can be used for the accurate profiling of DUB activity as well as the identification of DUBs and DUB inhibitor selectivity. Previously, active-site directed DUB probes have been obtained using intein-based methods that have inherent limitations. Total chemical synthesis of ubiquitin allows for easy incorporation of different tags, such as fluorescent reporters, affinity tags, and cleavable linkers. Here, we describe the total chemical synthesis of a fluorescent active-site directed DUB probe, which facilitates fast, in-gel detection of active DUBs and circumvents the use of Western blot analysis. In addition, an in-gel activity-based DUB profiling assay is described in detail, in which the fluorescent DUB probe is used to visualize active DUBs in cell lysates. Finally, an inhibition assay is described in which the fluorescent probe is used to determine the specificity and potency of a small molecule DUB inhibitor.

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 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
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. Glickman MH, Ciechanover A (2002) The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev 82(2):373–428. doi:10.1152/physrev.00027.2001

    Article  CAS  PubMed  Google Scholar 

  2. Reyes-Turcu FE, Ventii KH, Wilkinson KD (2009) Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes. Annu Rev Biochem 78:363–397. doi:10.1146/annurev.biochem.78.082307.091526

    Article  CAS  PubMed  Google Scholar 

  3. Edelmann MJ, Nicholson B, Kessler BM (2011) Pharmacological targets in the ubiquitin system offer new ways of treating cancer, neurodegenerative disorders and infectious diseases. Expert Rev Mol Med 13:e35. doi:10.1017/S1462399411002031

    Article  PubMed  PubMed Central  Google Scholar 

  4. Wilkinson KD (2009) DUBs at a glance. J Cell Sci 122(Pt 14):2325–2329. doi:10.1242/jcs.041046

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 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(7312):179–184. doi:10.1038/nature09299

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. D'Arcy P, Brnjic S, Olofsson MH, Fryknas M, Lindsten K, De Cesare M, Perego P, Sadeghi B, Hassan M, Larsson R, Linder S (2011) Inhibition of proteasome deubiquitinating activity as a new cancer therapy. Nat Med 17(12):1636–1640. doi:10.1038/nm.2536

    Article  PubMed  Google Scholar 

  7. Borodovsky A, Ovaa H, Kolli N, Gan-Erdene T, Wilkinson KD, Ploegh HL, Kessler BM (2002) Chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family. Chem Biol 9(10):1149–1159

    Article  CAS  PubMed  Google Scholar 

  8. de Jong A, Merkx R, Berlin I, Rodenko B, Wijdeven RH, El Atmioui D, Yalcin Z, Robson CN, Neefjes JJ, Ovaa H (2012) Ubiquitin-based probes prepared by total synthesis to profile the activity of deubiquitinating enzymes. Chembiochem 13(15):2251–2258. doi:10.1002/cbic.201200497

    Article  PubMed  PubMed Central  Google Scholar 

  9. Ekkebus R, van Kasteren SI, Kulathu Y, Scholten A, Berlin I, Geurink PP, de Jong A, Goerdayal S, Neefjes J, Heck AJ, Komander D, Ovaa H (2013) On terminal alkynes that can react with active-site cysteine nucleophiles in proteases. J Am Chem Soc 135(8):2867–2870. doi:10.1021/ja309802n

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Altun M, Kramer HB, Willems LI, McDermott JL, Leach CA, Goldenberg SJ, Kumar KG, Konietzny R, Fischer R, Kogan E, Mackeen MM, McGouran J, Khoronenkova SV, Parsons JL, Dianov GL, Nicholson B, Kessler BM (2011) Activity-based chemical proteomics accelerates inhibitor development for deubiquitylating enzymes. Chem Biol 18(11):1401–1412. doi:10.1016/j.chembiol.2011.08.018

    Article  CAS  PubMed  Google Scholar 

  11. Mulder MP, El Oualid F, ter Beek J, Ovaa H (2014) A native chemical ligation handle that enables the synthesis of advanced activity-based probes: diubiquitin as a case study. Chembiochem 15(7):946–949. doi:10.1002/cbic.201402012

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Dang LC, Melandri FD, Stein RL (1998) Kinetic and mechanistic studies on the hydrolysis of ubiquitin C-terminal 7-amido-4-methylcoumarin by deubiquitinating enzymes. Biochemistry 37(7):1868–1879. doi:10.1021/bi9723360

    Article  CAS  PubMed  Google Scholar 

  13. Hassiepen U, Eidhoff U, Meder G, Bulber JF, Hein A, Bodendorf U, Lorthiois E, Martoglio B (2007) A sensitive fluorescence intensity assay for deubiquitinating proteases using ubiquitin-rhodamine110-glycine as substrate. Anal Biochem 371(2):201–207. doi:10.1016/j.ab.2007.07.034

    Article  CAS  PubMed  Google Scholar 

  14. Frickel EM, Quesada V, Muething L, Gubbels MJ, Spooner E, Ploegh H, Artavanis-Tsakonas K (2007) Apicomplexan UCHL3 retains dual specificity for ubiquitin and Nedd8 throughout evolution. Cell Microbiol 9(6):1601–1610. doi:10.1111/j.1462-5822.2007.00896.x

    Article  CAS  PubMed  Google Scholar 

  15. Tirat A, Schilb A, Riou V, Leder L, Gerhartz B, Zimmermann J, Worpenberg S, Eidhoff U, Freuler F, Stettler T, Mayr L, Ottl J, Leuenberger B, Filipuzzi I (2005) Synthesis and characterization of fluorescent ubiquitin derivatives as highly sensitive substrates for the deubiquitinating enzymes UCH-L3 and USP-2. Anal Biochem 343(2):244–255. doi:10.1016/j.ab.2005.04.023

    Article  CAS  PubMed  Google Scholar 

  16. Geurink PP, El Oualid F, Jonker A, Hameed DS, Ovaa H (2012) A general chemical ligation approach towards isopeptide-linked ubiquitin and ubiquitin-like assay reagents. Chembiochem 13(2):293–297. doi:10.1002/cbic.201100706

    Article  CAS  PubMed  Google Scholar 

  17. Mevissen TE, Hospenthal MK, Geurink PP, Elliott PR, Akutsu M, Arnaudo N, Ekkebus R, Kulathu Y, Wauer T, El Oualid F, Freund SM, Ovaa H, Komander D (2013) OTU deubiquitinases reveal mechanisms of linkage specificity and enable ubiquitin chain restriction analysis. Cell 154(1):169–184. doi:10.1016/j.cell.2013.05.046

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Schneider T, Schneider D, Rosner D, Malhotra S, Mortensen F, Mayer TU, Scheffner M, Marx A (2014) Dissecting ubiquitin signaling with linkage-defined and protease resistant ubiquitin chains. Angew Chem Int Ed Engl 53(47):12925–12929. doi:10.1002/anie.201407192

    Article  CAS  PubMed  Google Scholar 

  19. Shanmugham A, Fish A, Luna-Vargas MP, Faesen AC, El Oualid F, Sixma TK, Ovaa H (2010) Nonhydrolyzable ubiquitin-isopeptide isosteres as deubiquitinating enzyme probes. J Am Chem Soc 132(26):8834–8835. doi:10.1021/ja101803s

    Article  CAS  PubMed  Google Scholar 

  20. Lam YA, Xu W, DeMartino GN, Cohen RE (1997) Editing of ubiquitin conjugates by an isopeptidase in the 26S proteasome. Nature 385(6618):737–740. doi:10.1038/385737a0

    Article  CAS  PubMed  Google Scholar 

  21. Licchesi JD, Mieszczanek J, Mevissen TE, Rutherford TJ, Akutsu M, Virdee S, El Oualid F, Chin JW, Ovaa H, Bienz M, Komander D (2012) An ankyrin-repeat ubiquitin-binding domain determines TRABID’s specificity for atypical ubiquitin chains. Nat Struct Mol Biol 19(1):62–71. doi:10.1038/nsmb.2169

    Article  CAS  Google Scholar 

  22. Wang J, Loveland AN, Kattenhorn LM, Ploegh HL, Gibson W (2006) High-molecular-weight protein (pUL48) of human cytomegalovirus is a competent deubiquitinating protease: mutant viruses altered in its active-site cysteine or histidine are viable. J Virol 80(12):6003–6012. doi:10.1128/JVI.00401-06

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. White RR, Miyata S, Papa E, Spooner E, Gounaris K, Selkirk ME, Artavanis-Tsakonas K (2011) Characterisation of the Trichinella spiralis deubiquitinating enzyme, TsUCH37, an evolutionarily conserved proteasome interaction partner. PLoS Negl Trop Dis 5(10):e1340. doi:10.1371/journal.pntd.0001340

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. 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(18):5187–5196. doi:10.1093/emboj/20.18.5187

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Ovaa H, Galardy PJ, Ploegh HL (2005) Mechanism-based proteomics tools based on ubiquitin and ubiquitin-like proteins: synthesis of active site-directed probes. Methods Enzymol 399:468–478. doi:10.1016/S0076-6879(05)99032-0

    Article  CAS  PubMed  Google Scholar 

  26. Virdee S, Ye Y, Nguyen DP, Komander D, Chin JW (2010) Engineered diubiquitin synthesis reveals Lys29-isopeptide specificity of an OTU deubiquitinase. Nat Chem Biol 6(10):750–757. doi:10.1038/nchembio.426

    Article  CAS  PubMed  Google Scholar 

  27. El Oualid F, Merkx R, Ekkebus R, Hameed DS, Smit JJ, de Jong A, Hilkmann H, Sixma TK, Ovaa H (2010) Chemical synthesis of ubiquitin, ubiquitin-based probes, and diubiquitin. Angew Chem Int Ed Engl 49(52):10149–10153. doi:10.1002/anie.201005995

    Article  PubMed  PubMed Central  Google Scholar 

  28. Kapuria V, Peterson LF, Fang D, Bornmann WG, Talpaz M, Donato NJ (2010) Deubiquitinase inhibition by small-molecule WP1130 triggers aggresome formation and tumor cell apoptosis. Cancer Res 70(22):9265–9276. doi:10.1158/0008-5472.CAN-10-1530

    Article  CAS  PubMed  Google Scholar 

  29. Sun H, Kapuria V, Peterson LF, Fang D, Bornmann WG, Bartholomeusz G, Talpaz M, Donato NJ (2011) Bcr-Abl ubiquitination and Usp9x inhibition block kinase signaling and promote CML cell apoptosis. Blood 117(11):3151–3162. doi:10.1182/blood-2010-03-276477

    Article  CAS  PubMed  Google Scholar 

  30. Liu S, Hanzlik RP (1992) Structure-activity relationships for inhibition of papain by peptide Michael acceptors. J Med Chem 35(6):1067–1075

    Article  CAS  PubMed  Google Scholar 

  31. Kvach MV, Stepanova IA, Prokhorenko IA, Stupak AP, Bolibrukh DA, Korshun VA, Shmanai VV (2009) Practical synthesis of isomerically pure 5- and 6-carboxytetramethylrhodamines, useful dyes for DNA probes. Bioconjug Chem 20(8):1673–1682. doi:10.1021/bc900037b

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Author notes

    Authors and Affiliations

    1. Department of Chemical Immunology, Leiden University Medical Center, Albinusdreef 2, 2333, ZA, Leiden, The Netherlands

      Yves Leestemaker & Huib Ovaa

    2. Division of Cell Biology II, Netherlands Cancer Institute, Plesmanlaan 121, 1066, CX, Amsterdam, The Netherlands

      Yves Leestemaker, Annemieke de Jong & Huib Ovaa

    Authors
    1. Yves Leestemaker
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Annemieke de Jong
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Huib Ovaa
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to Huib Ovaa .

    Editor information

    Editors and Affiliations

    1. Bio-Organic Synthesis Group Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands

      Herman S. Overkleeft

    2. Bio-Organic Synthesis Group Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands

      Bogdan I. Florea

    Rights and permissions

    Reprints and permissions

    Copyright information

    © 2017 Springer Science+Business Media New York

    About this protocol

    Cite this protocol

    Leestemaker, Y., de Jong, A., Ovaa, H. (2017). Profiling the Activity of Deubiquitinating Enzymes Using Chemically Synthesized Ubiquitin-Based Probes. In: Overkleeft, H., Florea, B. (eds) Activity-Based Proteomics. Methods in Molecular Biology, vol 1491. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6439-0_9

    Download citation

    • DOI: https://doi.org/10.1007/978-1-4939-6439-0_9

    • Published:

    • Publisher Name: Humana Press, New York, NY

    • Print ISBN: 978-1-4939-6437-6

    • Online ISBN: 978-1-4939-6439-0

    • eBook Packages: Springer Protocols

    Publish with us

    Policies and ethics

    Search

    Navigation