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  • Deubiquitinating enzyme amino acid profiling reveals a class of ubiquitin esterases.

Deubiquitinating enzyme amino acid profiling reveals a class of ubiquitin esterases.

Proceedings of the National Academy of Sciences of the United States of America (2021-01-23)
Virginia De Cesare, Daniel Carbajo Lopez, Peter D Mabbitt, Adam J Fletcher, Mathieu Soetens, Odetta Antico, Nicola T Wood, Satpal Virdee
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Roche
cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail, Protease Inhibitor Cocktail Tablets provided in a glass vial, Tablets provided in a glass vial
Sigma-Aldrich
MG-132 - CAS 133407-82-6 - Calbiochem, A cell-permeable, potent, reversible proteasome inhibitor (Ki = 4 nM).
Sigma-Aldrich
Ammonium citrate dibasic, puriss., ≥98%
Sigma-Aldrich
Dihydroxyacetone phosphate lithium salt, ≥95.0% (TLC)