Deubiquitinating enzyme amino acid profiling reveals a class of ubiquitin esterases

Virginia De Cesare, Daniel Carbajo Lopez, Peter D. Mabbitt, Adam J. Fletcher, Mathieu Soetens, Odetta Antico, Nicola T. Wood, Satpal Virdee (Lead / Corresponding author)

Research output: Contribution to journalArticlepeer-review

41 Citations (Scopus)
154 Downloads (Pure)


The reversibility of ubiquitination by the action of deubiquitinating enzymes (DUBs) serves as an important regulatory layer within the ubiquitin system. Approximately 100 DUBs are encoded by the human genome, and many have been implicated with pathologies, including neurodegeneration and cancer. Non-lysine ubiquitination is chemically distinct, and its physiological importance is emerging. Here, we couple chemically and chemoenzymatically synthesized ubiquitinated lysine and threonine model substrates to a mass spectrometry-based DUB assay. Using this platform, we profile two-thirds of known catalytically active DUBs for threonine esterase and lysine isopeptidase activity and find that most DUBs demonstrate dual selectivity. However, with two anomalous exceptions, the ovarian tumor domain DUB class demonstrates specific (iso)peptidase activity. Strikingly, we find the Machado-Joseph disease (MJD) class to be unappreciated non-lysine DUBs with highly specific ubiquitin esterase activity rivaling the efficiency of the most active isopeptidases. Esterase activity is dependent on the canonical catalytic triad, but proximal hydrophobic residues appear to be general determinants of non-lysine activity. Our findings also suggest that ubiquitin esters have appreciable cellular stability and that non-lysine ubiquitination is an integral component of the ubiquitin system. Its regulatory sophistication is likely to rival that of canonical ubiquitination.

Original languageEnglish
Article numbere2006947118
Number of pages12
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number4
Early online date21 Jan 2021
Publication statusPublished - 26 Jan 2021


  • ubiquitin
  • DUBs
  • non-lysine ubiquitination
  • Ubiquitin
  • Non-lysine ubiquitination

ASJC Scopus subject areas

  • General


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