Comprehensive Landscape of Active Deubiquitinating Enzymes Profiled by Advanced Chemoproteomics

Abstract

Enzymes that bind and process ubiquitin, a small 76-amino-acid protein, have been recognized as pharmacological targets in oncology, immunological disorders, and neurodegeneration. Mass spectrometry technology has now reached the capacity to cover the proteome with enough depth to interrogate entire biochemical pathways including those that contain DUBs and E3 ligase substrates. We have recently characterized the breast cancer cell (MCF7) deep proteome by detecting and quantifying ~10,000 proteins, and within this data set, we can detect endogenous expression of 65 deubiquitylating enzymes (DUBs), whereas matching transcriptomics detected 78 DUB mRNAs. Since enzyme activity provides another meaningful layer of information in addition to the expression levels, we have combined advanced mass spectrometry technology, pre-fractionation, and more potent/selective ubiquitin active-site probes with propargylic-based electrophiles to profile 74 DUBs including distinguishable isoforms for 5 DUBs in MCF7 crude extract material. Competition experiments with cysteine alkylating agents and pan-DUB inhibitors combined with probe labeling revealed the proportion of active cellular DUBs directly engaged with probes by label-free quantitative (LFQ) mass spectrometry. This demonstrated that USP13, 39, and 40 are non-reactive to probe, indicating restricted enzymatic activity under these cellular conditions. Our extended chemoproteomics workflow increases depth of covering the active DUBome, including isoform-specific resolution, and provides the framework for more comprehensive cell-based small-molecule DUB selectivity profiling.

Report of the first patient with a homozygous OTUD7A variant responsible for epileptic encephalopathy and related proteasome dysfunction

“…This gene encodes for the OTU Domain‐containing protein 7A belonging to a protein family involved in the regulation many cellular processes via deubiquitination, expressed in brain and localized to dendritic and synaptic compartments during the maturation of cortical neurons . Ubiquitin is a conserved, globular protein consisting of 76 amino acids that can be attached to proteins either in a mono or polymerized form, impacting on their activity, localization, interactome, and turnover . Ubiquitination is a reversible posttranslational modification, mediated by deubiquitinating enzymes, playing also a critical role in synapse formation and function .…”

“…impacting on their activity, localization, interactome, and turnover. 25 Ubiquitination is a reversible posttranslational modification, mediated by deubiquitinating enzymes, playing also a critical role in synapse formation and function. 26 Pathogenic variants in deubiquitinases such as USP9X, USP27X and OTUD6B have been already associated with recessive human diseases responsible for mental retardation (MIM 300919, 300 984 and 617 452, respectively).…”

Strategies to Target Specific Components of the Ubiquitin Conjugation/Deconjugation Machinery

“…Application of this methodology to conjugate probes could enable the development of cellpermeable versions. Additionally, large scale biological screens combined with the latest chemoproteomic methods, similar to those carried out using monoubiquitin probes, could provide a more resolved picture of DUB activity using these more specific probes (Hewings et al, 2018;Pinto-Fernández et al, 2019). Overall, the expansion and combination of methods reviewed herein could open further possibilities, ultimately affording a panel of probes capable of targeting specific subsets or even individual enzymes.…”

“…Recently, large biological screens using these probes have aided in the development of a new chemoproteomic method that could potentially be used to identify the labeling site of any covalent modifier (Hewings et al, 2018). A similar screen demonstrated how chemoproteomics can be used to study DUBs in a more comprehensive manner (Pinto-Fernández et al, 2019). These examples elegantly demonstrate the depth of knowledge that can be obtained using these probes.…”

Deep analysis of the USP18-dependent ISGylome and proteome unveils important roles for USP18 in tumour cell antigenicity and radiosensitivity

“…10 % of the eluates was used for immunoblotting with the indicated antibodies. The remaining eluate was prepared for MS analysis as previously described 69,70 . Briefly: immunoprecipitated sample eluates were diluted to 175 μL with ultrapure water and reduced with 5 μL of DTT (200 mM in 0.1 M Tris, pH 7.8) for 30 min at 37 °C.…”