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A method for assaying deubiquitinating enzymes

Abstract

A general method for the assay of deubiquitinating enzymes was described in detail using 125I-labeled ubiquitin-fused αNH-MHISPPEPESEEEEEHYC (referred to as Ub-PESTc) as a substrate. Since the tyrosine residue in the PESTc portion of the fusion protein was almost exclusively radioiodinated under a mild labeling condition, such as using IODO-BEADS, the enzymes could be assayed directly by simple measurement of the radioactivity released into acid soluble products. Using this assay protocol, we could purify six deubiquitinating enzymes from chick skeletal muscle and yeast and compare their specific activities. Since the extracts of E. coli showed little or no activity against the substrate, the assay protocol should be useful for identification and purification of eukaryotic deubiquitinating enzymes cloned and expressed in the cells.

References

  1. 1.

    Rechsteiner, M. 1987. Ubiquitin-mediated pathways for intracellular proteolysis. Annu. Rev. Cell Biol. 3, 1–30.

    PubMed  Article  CAS  Google Scholar 

  2. 2.

    Hershko, A., and Ciechanover, A. 1992. The ubiquitin system for protein degradation. Annu. Rev. Biochem. 61, 761–807.

    PubMed  Article  CAS  Google Scholar 

  3. 3.

    Jentsch, S. 1992. The ubiquitin-conjugating system. Annu. Rev. Genet. 26, 179–207.

    PubMed  Article  CAS  Google Scholar 

  4. 4.

    Hochstrasser, M. 1996. Ubiquitin-dependent protein degradation. Annu. Rev. Genet. 30, 405–439.

    PubMed  Article  CAS  Google Scholar 

  5. 5.

    Wilkinson, K.D. 1995. Roles of ubiquitinylation in proteolysis and cellular regulation. Annu. Rev. Nutr. 15, 161–189.

    PubMed  Article  CAS  Google Scholar 

  6. 6.

    Carlson, N., and Rechsteiner, M. 1987. Microinjection of ubiquitin: Intracellular distribution of and metabolism in HeLa cells maintained under normal physiological conditions. J. Cell Biol. 104, 537–546.

    PubMed  Article  CAS  Google Scholar 

  7. 7.

    Carlson, N., Rogers, S., and Rechsteiner, M. 1987. Microinjection of ubiquitin: Changes in protein degradation in HeLa cells subjected to heat-shock. J. Cell Biol. 104, 547–555.

    PubMed  Article  CAS  Google Scholar 

  8. 8.

    Haas, A.L. 1988. Immunochemical probes of Ub pool dynamics. In Ubiquitin (Rechsteiner, M., ed.) pp. 173–206, Plenum Press, New York.

    Google Scholar 

  9. 9.

    Ozkaynak, E., Finley, D., and Varshavsky, A. 1984. The yeast ubiquitin gene: Head-to-tail repeats encoding a polyubiquitin precursor protein. Nature 312, 663–666.

    PubMed  Article  CAS  Google Scholar 

  10. 10.

    Lund, P.K., Moasts-Staats, B.M., Simmons, J.G., Hoyt, E., D’Ercole, A.J., Martin, F., and Van Wyk, J.J. 1985. Nucleotide sequence analysis of a cDNA encoding human ubiquitin reveals that ubiquitin is synthesized as a precursor. J. Biol. Chem. 260, 7609–7613.

    PubMed  CAS  Google Scholar 

  11. 11.

    Finley, D., Bartel, B., and Varshavsky, A. 1989. The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis. Nature 338, 394–401.

    PubMed  Article  CAS  Google Scholar 

  12. 12.

    Wilkinson, K.D. 1997. Regulation of ubiquitin-dependent processes by deubiquitinating enzymes. FASEB J. 11, 1245–1256.

    PubMed  CAS  Google Scholar 

  13. 13.

    Woo, S.K., Lee, J.I., Park, I.K., Yoo, Y.J., Cho, J.M., Tanaka, K., Kang, M.S., Ha, D.B. and Chung, C.H. 1995. Multiple ubiquitin C-terminal hydrolases from chick skeletal muscle. J. Biol. Chem. 270, 18766–18773.

    PubMed  Article  CAS  Google Scholar 

  14. 14.

    Chung, C.H., Woo, S.K., Lee, J.I., Park, I.K., Kang, M.S. and Ha, D.B. 1996. Ubiquitin Cterminal hydrolases in chick skeletal muscle. Adv. Exp. Med. Biol. 384, 203–208.

    Google Scholar 

  15. 15.

    Baek, S.H., Choi, K.S., Yoo, Y.J., Cho, J.M., Baker, R.T., Tanaka, K. and Chung, C.H. 1997. Molecular cloning of a novel ubiquitin-specific protease, UBP41, with isopeptidase activity in chick skeletal muscle. J. Biol. Chem. 272, 25560–25565.

    PubMed  Article  CAS  Google Scholar 

  16. 16.

    Woo, S.K., Baek, S.H., Lee, J.I., Yoo, Y.J., Cho, J.M., Kang, M.S. and Chung, C.H. 1997. Purification and characterization of a new ubiquitin C-terminal hydrolase (UCH-1) with isopeptidase activity from chick skeletal muscle. J. Biochem. 121, 684–689.

    PubMed  CAS  Google Scholar 

  17. 17.

    Baek, S.H., Woo, S.K., Lee, J.I., Yoo, Y.J., Cho, J.M., Kang, M.S., Tanaka, K. and Chung, C.H. 1997. New de-ubiquitinating enzyme, ubiquitin C-terminal hydrolase 8, in chick skeletal muscle. Biochem. J. 325, 325–330.

    PubMed  CAS  Google Scholar 

  18. 18.

    Park, K.C., Woo, S.K., Yoo, Y.J., Wyndham, A.M., Baker, R.T. and Chung, C.H. 1997. Purification and characterization of UBP6, a new ubiquitin specific protease in Saccharomyces cerevisiae. Arch. Biochem. Biophys. 347, 78–84.

    PubMed  Article  CAS  Google Scholar 

  19. 19.

    Stein, R.L., Chen, Z., and Melandri, F. 1995. Kinetic studies of isopeptidase T: Modulation of peptidase activity by ubiquitin. Biochemistry 34, 12616–12623.

    PubMed  Article  CAS  Google Scholar 

  20. 20.

    Miller, H.I., Henzel, W.J., Ridgway, J.B., Kuang, W.J., Chisholm, V., and Liu, C.C. 1989. Cloning and expression of a yeast ubiquitin-protein cleaving activity in E. coli. Bio/Technology 7, 698–704.

    Article  CAS  Google Scholar 

  21. 21.

    Yoo, Y., Rote, K., and Rechsteiner, M. 1989. Synthesis of peptides as cloned ubiquitin extensions. J. Biol. Chem. 264, 17078–17083.

    PubMed  CAS  Google Scholar 

  22. 22.

    Markwell, M.A.K. 1982. A new solid-phase reagent to iodinate proteins. Anal. Biochem. 125, 427–435.

    PubMed  Article  CAS  Google Scholar 

  23. 23.

    Yoo, S.J., Seol, J.H., Shin, D.H., Rohrwild, M., Kang, M.S., Tanaka, K., Goldberg, A.L., and Chung, C.H. 1996. Purification and characterization of the heat shock proteins HslV and HslU that forms a new ATP-dependent protease in Escherichia coli. J. Biol. Chem. 271, 14035–14040.

    PubMed  Article  CAS  Google Scholar 

  24. 24.

    Bradford, M.M. A rapid and sensitive method of quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.

  25. 25.

    Schägger, H., and von Jagow, G. 1987. Tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368–379.

    PubMed  Article  Google Scholar 

  26. 26.

    Hershko, A., and Rose, I.A. 1977. Ubiquitin-aldehyde: a general inhibitor of ubiquitin-protein ligase system. Proc. Natl. Acad. Sci. U.S.A. 84, 1829–1833.

    Article  Google Scholar 

  27. 27.

    Wilkinson, K.D., Tashayev, V.L., O’Connor, L.B., Larsen, C.N., Kasperek, E., and Pickart, C.M. 1995. Metabolism of the polyubiquitin degradation signal: structure, mechanism, and role of isopeptidase T. Biochemistry 34, 14535–14546.

    PubMed  Article  CAS  Google Scholar 

  28. 28.

    Baek, S.H., and Chung, C.H. manuscript in preparation.

  29. 29.

    Dang, L.C., Melandri, F.D., and Stein, R.L. 1998. Kinetic and mechanistic studies on the hydrolysis of ubiquitin C-terminal 7-amido-4-methylcoumarin by deubiquitinating enzymes. Biochemistry 37, 1868–1879.

    PubMed  Article  CAS  Google Scholar 

  30. 30.

    Wilkinson, K.D., Deshpande, S., and Larsen, C.N. 1992. Comparisons of neuronal (PGP 9.5) and non-neuronal ubiquitin C-terminal hydrolases. Biochem. Soc. Trans. 20, 631–636.

    PubMed  CAS  Google Scholar 

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Correspondence to Jae Il Lee or Chin Ha Chung.

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Lee, J.I., Woo, S.K., Kim, K.I. et al. A method for assaying deubiquitinating enzymes. Biol Proced Online 1, 92–99 (1998). https://doi.org/10.1251/bpo11

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Keywords

  • Assay Protocol
  • Biological Procedure
  • Strain AR13
  • Deubiquitinating Enzyme
  • Biological Procedure Online