Chemically synthesized ubiquitin extension proteins detect distinct catalytic capacities of deubiquitinating enzymes

Robert Layfield, Kate Franklin, Michael Landon, Gail Walker, Pu Wang, Robert Ramage, Angus Brown, Steven Love, Kirstie Urquhart, Thomas Muir, Rohan Baker, R. John Mayer

Research output: Contribution to journalArticle

30 Scopus citations

Abstract

We have used solid-phase chemistry to synthesize proteins equivalent to a human ubiquitin precursor (ubiquitin-52-amino-acid ribosomal protein fusion; UBICEP52) and representative of isopeptide-linked ubiquitin-protein conjugates [ubiquitin-(εN)-lysine]; these proteins were precisely cleaved by a purified recombinant Drosophila deubiquitinating enzyme (DUB), UCH-D. Along with the previously synthesized ubiquitin-(αN)-valine, these synthetic proteins were used as substrates to assess the catalytic capacities of a number of diverse DUBs expressed in Escherichia coli: human HAUSP; mouse Unp; and yeast Ubps 1p, 2p, 3p, 6p, 11p, and 15p and Yuh1p. Distinct specificities of these enzymes were detected; notably, in addition to UCH-D, isopeptidase activity [ubiquitin-(εN)-lysine cleavage] was only associated with Yuh1p, Unp, Ubp1p, and Ubp2p. Additionally, human placental 26S proteasomes were only able to cleave UBICEP52 and ubiquitin-(εN)-lysine, suggesting that 26S proteasome-associated DUBs are class II-like. This work demonstrates that the synthetic approach offers an alternative to recombinant methods for the production of small proteins in vitro.

Original languageEnglish (US)
Pages (from-to)40-49
Number of pages10
JournalAnalytical Biochemistry
Volume274
Issue number1
DOIs
StatePublished - Oct 1 1999
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Layfield, R., Franklin, K., Landon, M., Walker, G., Wang, P., Ramage, R., Brown, A., Love, S., Urquhart, K., Muir, T., Baker, R., & Mayer, R. J. (1999). Chemically synthesized ubiquitin extension proteins detect distinct catalytic capacities of deubiquitinating enzymes. Analytical Biochemistry, 274(1), 40-49. https://doi.org/10.1006/abio.1999.4234