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

doi:10.1073/pnas.2006947118

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.

Highlights

Major histocompatibility complex class I (MHC-I) molecules [8, 9]
We tested the activity of 53 deubiquitinating enzymes (DUBs) against both lysine and non-lysine ubiquitinated model substrates, making the current work the most extensive and cross-validated study on non-lysine DUB activity to date
On the whole, ubiquitin-specific protease (USP) and ubiquitin C-terminal hydrolase (UCH) class DUBs mediate both isopeptidase and esterase activity with comparable kinetics

Summary

Results

To determine DUB activity and specificity toward either ester or isopeptide bonds, we employed a previously developed MALDI-TOF DUB assay [17] and the model substrates Ub-Lysine (Ub-Lys) and UbThreonine (Ub-Thr) (Fig. 1 A and B). In contrast to USP and UCH classes, OTU family members invariably demonstrate efficient lysine isopeptidase activity toward our model substrate, but negligible threonine esterase activity (Fig. 2B). With the exception of ATXN3L, all MJD DUBs demonstrate preferential threonine esterase activity (Fig. 2B) This is notable for JOSD1 where isopeptidase activity is negligible but quantitative cleavage of the Ub-Thr substrate is observed after the first time point. Consistent with activity of these DUBs being dependent on polyUb linkage context, negligible activity was observed toward either of our model isopeptide or ester-linked substrates (Fig. 2B). OTUB1 OTUB2 YOD1 vOTU OTUD5 OTUD1 OTUD3 OTUD6B OTUD6A OTULIN VCPIP1 ZRANB1 A20 OTUD7B OTUD7A

A20 CEZANNE

Discussion

C Catalytic

Materials and Methods