Deubiquitinating Enzyme Amino Acid Profiling Reveals a Class of Ubiquitin Esterases

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 matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) 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 (OTU) DUB class demonstrate specific (iso)peptidase activity. Strikingly, we find the Machado Josephin Domain (MJD) class to be unappreciated non-lysine DUBs with highly specific ubiquitin esterase activity that rivals 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. These findings suggest that non-lysine ubiquitination is an integral component of the ubiquitin system, with regulatory sophistication comparable to that of canonical ubiquitination.