SpringerLink
Log in

Diggin′ on U(biquitin): A Novel Method for the Identification of Physiological E3 Ubiquitin Ligase Substrates

  • Original Paper
  • Published:
Cell Biochemistry and Biophysics Aims and scope Submit manuscript

Abstract

The ubiquitin–proteasome system (UPS) plays a central role in maintaining protein homeostasis, emphasized by a myriad of diseases that are associated with altered UPS function such as cancer, muscle-wasting, and neurodegeneration. Protein ubiquitination plays a central role in both the promotion of proteasomal degradation as well as cellular signaling through regulation of the stability of transcription factors and other signaling molecules. Substrate-specificity is a critical regulatory step of ubiquitination and is mediated by ubiquitin ligases. Recent studies implicate ubiquitin ligases in multiple models of cardiac diseases such as cardiac hypertrophy, atrophy, and ischemia/reperfusion injury, both in a cardioprotective and maladaptive role. Therefore, identifying physiological substrates of cardiac ubiquitin ligases provides both mechanistic insights into heart disease as well as possible therapeutic targets. Current methods identifying substrates for ubiquitin ligases rely heavily upon non-physiologic in vitro methods, impeding the unbiased discovery of physiological substrates in relevant model systems. Here we describe a novel method for identifying ubiquitin ligase substrates utilizing tandem ubiquitin binding entities technology, two-dimensional differential in gel electrophoresis, and mass spectrometry, validated by the identification of both known and novel physiological substrates of the ubiquitin ligase MuRF1 in primary cardiomyocytes. This method can be applied to any ubiquitin ligase, both in normal and disease model systems, in order to identify relevant physiological substrates under various biological conditions, opening the door to a clearer mechanistic understanding of ubiquitin ligase function and broadening their potential as therapeutic targets.

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

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price includes VAT (Germany)

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Colland, F. (2010). The therapeutic potential of deubiquitinating enzyme inhibitors. Biochemical Society Transactions, 38(Pt 1), 137–143.

    Article  PubMed  CAS  Google Scholar 

  2. Lim, K. H., & Baek, K. H. (2013). Deubiquitinating enzymes as therapeutic targets in cancer. Current Pharmaceutical Design, 19(22), 4039–4052.

    Article  PubMed  CAS  Google Scholar 

  3. Ikeda, F., & Dikic, I. (2008). Atypical ubiquitin chains: new molecular signals. “Protein modifications: beyond the usual suspects” review series. EMBO Reports, 9(6), 536–542.

    Article  PubMed  CAS  Google Scholar 

  4. Edelmann, M. J., Nicholson, B., & Kessler, B. M. (2011). Pharmacological targets in the ubiquitin system offer new ways of treating cancer, neurodegenerative disorders and infectious diseases. Expert Reviews in Molecular Medicine, 13, e35.

    Article  PubMed  Google Scholar 

  5. Zolk, O., Schenke, C., & Sarikas, A. (2006). The ubiquitin-proteasome system: focus on the heart. Cardiovascular Research, 70(3), 410–421.

    Article  PubMed  CAS  Google Scholar 

  6. Wilkie, Neil and Davies S. Drug Discovery World, Drug Discovery and Development News.

  7. Centner, T., Yano, J., Kimura, E., McElhinny, A. S., Pelin, K., Witt, C. C., et al. (2001). Identification of muscle specific ring finger proteins as potential regulators of the titin kinase domain. Journal of Molecular Biology, 306(4), 717–726.

    Article  PubMed  CAS  Google Scholar 

  8. Spencer, J. A., Eliazer, S., Ilaria, R. L., Richardson, J. A., & Olson, E. N. (2000). Regulation of microtubule dynamics and myogenic differentiation by MURF, a striated muscle RING-finger protein. The Journal of cell biology, 150(4), 771–784.

    Article  PubMed  CAS  Google Scholar 

  9. Kedar, V., McDonough, H., Arya, R., Li, H–. H., Rockman, H. A., & Patterson, C. (2004). Muscle-specific RING finger 1 is a bona fide ubiquitin ligase that degrades cardiac troponin I. Proceedings of the National Academy of Sciences of the United States of America, 101(52), 18135–18140.

    Article  PubMed  CAS  Google Scholar 

  10. Polge, C., Heng, A.-E., Jarzaguet, M., Ventadour, S., Claustre, A., Combaret, L., et al. (2011). Muscle actin is polyubiquitinylated in vitro and in vivo and targeted for breakdown by the E3 ligase MuRF1. FASEB Journal, 25(11), 3790–3802.

    Article  PubMed  CAS  Google Scholar 

  11. Willis, M. S., Zungu, M., & Patterson, C. (2010). Cardiac muscle ring finger-1–friend or foe? Trends in Cardiovascular Medicine, 20(1), 12–16.

    Google Scholar 

  12. Mearini, G., Schlossarek, S., Willis, M. S., & Carrier, L. (2008). The ubiquitin-proteasome system in cardiac dysfunction. Biochimica et Biophysica Acta, 1782(12), 749–763.

    Article  PubMed  CAS  Google Scholar 

  13. Powell, S. R., Herrmann, J., Lerman, A., Patterson, C., & Wang, X. (2012). The ubiquitin-proteasome system and cardiovascular disease. Progress in Molecular Biology and Translational Science, 109, 295–346.

    Article  PubMed  CAS  Google Scholar 

  14. Powell, S. R. (2006). The ubiquitin-proteasome system in cardiac physiology and pathology. American Journal of Physiology - Heart and Circulatory Physiology, 291(1), H1–H19.

    Google Scholar 

  15. Zhang, Y., Zeng, Y., Wang, M., Tian, C., Ma, X., Chen, H., et al. (2011). Cardiac-specific overexpression of E3 ligase Nrdp1 increases ischemia and reperfusion-induced cardiac injury. Basic Research in Cardiology, 106(3), 371–383.

    Article  PubMed  CAS  Google Scholar 

  16. Li, H–. H., Du, J., Fan, Y.-N., Zhang, M.-L., Liu, D.-P., Li, L., et al. (2011). The ubiquitin ligase MuRF1 protects against cardiac ischemia/reperfusion injury by its proteasome-dependent degradation of phospho-c-Jun. The American journal of pathology, 178(3), 1043–1058.

    Article  PubMed  CAS  Google Scholar 

  17. Yu, X., & Kem, D. C. (2010). Proteasome inhibition during myocardial infarction. Cardiovascular Research, 85(2), 312–320.

    Article  PubMed  CAS  Google Scholar 

  18. Nowis, D., Maczewski, M., Mackiewicz, U., Kujawa, M., Ratajska, A., Wieckowski, M. R., et al. (2010). Cardiotoxicity of the anticancer therapeutic agent bortezomib. The American Journal of Pathology, 176(6), 2658–2668.

    Article  PubMed  CAS  Google Scholar 

  19. Deshaies, R. J., & Joazeiro, C. A. P. (2009). RING domain E3 ubiquitin ligases. Annual Review of Biochemistry, 78, 399–434.

    Article  PubMed  CAS  Google Scholar 

  20. Wu, Z., Chen, Y., Yang, T., Gao, Q., Yuan, M., & Ma, L. (2012). Targeted ubiquitination and degradation of G-protein-coupled receptor kinase 5 by the DDB1-CUL4 ubiquitin ligase complex. PLoS One, 7(8), e43997.

  21. Peschiaroli, A., Dorrello, N. V., Guardavaccaro, D., Venere, M., Halazonetis, T., Sherman, N. E., et al. (2006). SCFbetaTrCP-mediated degradation of Claspin regulates recovery from the DNA replication checkpoint response. Molecular Cell, 23(3), 319–329.

    Article  PubMed  CAS  Google Scholar 

  22. Kus, B., Gajadhar, A., Stanger, K., Cho, R., Sun, W., Rouleau, N., et al. (2005). A high throughput screen to identify substrates for the ubiquitin ligase Rsp5. The Journal of Biological Chemistry, 280(33), 29470–29478.

    Article  PubMed  CAS  Google Scholar 

  23. Loch, C. M., Eddins, M. J., & Strickler, J. E. (2011). Protein microarrays for the identification of praja1 e3 ubiquitin ligase substrates. Cell Biochemistry and Biophysics, 60(1–2), 127–135.

    Article  PubMed  CAS  Google Scholar 

  24. Qian, S.-B., McDonough, H., Boellmann, F., Cyr, D. M., & Patterson, C. (2006). CHIP-mediated stress recovery by sequential ubiquitination of substrates and Hsp70. Nature, 440(7083), 551–555.

    Article  PubMed  CAS  Google Scholar 

  25. Hjerpe, R., Aillet, F., Lopitz-Otsoa, F., Lang, V., England, P., & Rodriguez, M. S. (2009). Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities. EMBO Reports, 10(11), 1250–1258.

    Article  PubMed  CAS  Google Scholar 

  26. Toraason, M., Luken, M. E., Breitenstein, M., Krueger, J. A., & Biagini, R. E. (1989). Comparative toxicity of allylamine and acrolein in cultured myocytes and fibroblasts from neonatal rat heart. Toxicology, 56(1), 107–117.

    Article  PubMed  CAS  Google Scholar 

  27. Osorio, C., Sullivan, P. M., He, D. N., Mace, B. E., Ervin, J. F., Strittmatter, W. J., et al. (2007). Mortalin is regulated by APOE in hippocampus of AD patients and by human APOE in TR mice. Neurobiology of Aging, 28(12), 1853–1862.

    Article  PubMed  CAS  Google Scholar 

  28. Jiang, J., Ballinger, C. A., Wu, Y., Dai, Q., Cyr, D. M., Höhfeld, J., et al. (2001). CHIP is a U-box-dependent E3 ubiquitin ligase: identification of Hsc70 as a target for ubiquitylation. The Journal of Biological Chemistry, 276(46), 42938–42944.

    Article  PubMed  CAS  Google Scholar 

  29. Willis, M. S., Schisler, J. C., Li, L., Rodríguez, J. E., Hilliard, E. G., Charles, P. C., et al. (2009). Cardiac muscle ring finger-1 increases susceptibility to heart failure in vivo. Circulation Research, 105(1), 80–88.

    Article  PubMed  CAS  Google Scholar 

  30. Witt, S. H., Granzier, H., Witt, C. C., & Labeit, S. (2005). MURF-1 and MURF-2 target a specific subset of myofibrillar proteins redundantly: towards understanding MURF-dependent muscle ubiquitination. Journal of Molecular Biology, 350(4), 713–722.

    Article  PubMed  CAS  Google Scholar 

  31. Kim, W., Bennett, E. J., Huttlin, E. L., Guo, A., Li, J., Possemato, A., et al. (2011). Systematic and quantitative assessment of the ubiquitin-modified proteome. Molecular Cell, 44(2), 325–340.

    Article  PubMed  CAS  Google Scholar 

  32. Cohen, S., Brault, J. J., Gygi, S. P., Glass, D. J., Valenzuela, D. M., Gartner, C., et al. (2009). During muscle atrophy, thick, but not thin, filament components are degraded by MuRF1-dependent ubiquitylation. The Journal of Cell Biology, 185(6), 1083–1095.

    Article  PubMed  CAS  Google Scholar 

  33. Wagner, S. A., Beli, P., Weinert, B. T., Nielsen, M. L., Cox, J., Mann, M., et al. (2011). A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles. Molecular and Cellular Proteomics, 10(10), M111.013284.

  34. Portbury, A. L., Willis, M. S., & Patterson, C. (2011). Tearin’ up my heart: proteolysis in the cardiac sarcomere. The Journal of Biological Chemistry, 286(12), 9929–9934.

    Article  PubMed  CAS  Google Scholar 

  35. David, Y., Ternette, N., Edelmann, M. J., Ziv, T., Gayer, B., Sertchook, R., et al. (2011). E3 ligases determine ubiquitination site and conjugate type by enforcing specificity on E2 enzymes. The Journal of Biological Chemistry, 286(51), 44104–44115.

    Article  PubMed  CAS  Google Scholar 

  36. Hoeller, D., Hecker, C.-M., Wagner, S., Rogov, V., Dötsch, V., & Dikic, I. (2007). E3-independent monoubiquitination of ubiquitin-binding proteins. Molecular Cell, 26(6), 891–898.

    Article  PubMed  CAS  Google Scholar 

  37. Friedman, D. B., Hoving, S., & Westermeier, R. (2009). Isoelectric focusing and two-dimensional gel electrophoresis. Methods in Enzymology, 463, 515–540.

    Article  PubMed  CAS  Google Scholar 

  38. Zhu, W., Smith, J. W., & Huang, C.-M. (2010). Mass spectrometry-based label-free quantitative proteomics. Journal of Biomedicine and Biotechnology, 2010, 840518.

    PubMed  Google Scholar 

Download references

Acknowledgments

The authors would like to thank the Michael Hooker Proteomics Center at UNC for protein identification services and Andrea Portbury for her critical review of this manuscript.

Author information

Authors and Affiliations

  1. Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Carrie E. Rubel

  2. McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Carrie E. Rubel, Jonathan C. Schisler & Cam Patterson

  3. Division of Cardiology, University of North Carolina at Chapel Hill, 8200 Medical Biomolecular Research Building, Chapel Hill, NC, 27599-7126, USA

    Jonathan C. Schisler & Cam Patterson

  4. Program in Molecular Biology and Biotechnology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Eric D. Hamlett

  5. Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Robert M. DeKroon & Oscar Alzate

  6. Cardiovascular Division, BHF Centre of Research Excellence, King’s College London, London, UK

    Mathias Gautel

Authors
  1. Carrie E. Rubel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jonathan C. Schisler
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Eric D. Hamlett
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Robert M. DeKroon
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mathias Gautel
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Oscar Alzate
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Cam Patterson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Cam Patterson.

Additional information

Jonathan C. Schisler and Carrie E. Rubel contributed equally to this study.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rubel, C.E., Schisler, J.C., Hamlett, E.D. et al. Diggin′ on U(biquitin): A Novel Method for the Identification of Physiological E3 Ubiquitin Ligase Substrates. Cell Biochem Biophys 67, 127–138 (2013). https://doi.org/10.1007/s12013-013-9624-6

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12013-013-9624-6

Keywords

Search

Navigation