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Original Research ARTICLE Provisionally accepted The full-text will be published soon. Notify me

Front. Chem. | doi: 10.3389/fchem.2019.00592

Comprehensive landscape of active deubiquitinating enzymes profiled by advanced chemoproteomics

 Adán Pinto-Fernández1, 2*,  Simon Davis1, 2, Abigail B. Schofield1, 3,  Hannah C. Scott1, 2, Ping Zhang1, 4,  Eidarus Salah1, 5, 6,  Sebastian Mathea6, 7, Philip D. Charles1, 2,  Andreas Damianou1, 2, Gareth Bond1, 4,  Roman Fischer1, 2 and  Benedikt M. Kessler1, 2*
  • 1University of Oxford, United Kingdom
  • 2Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, United Kingdom
  • 3Christ Church, University of Oxford, United Kingdom
  • 4Ludwig Institute for Cancer Research, University of Oxford, United Kingdom
  • 5Department of Chemistry, University of Oxford, United Kingdom
  • 6Structural Genomics Consortium (United Kingdom), United Kingdom
  • 7Goethe University Frankfurt, Germany

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 of 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 five DUBs in MCF7 crude extract material. Competition experiments with cysteine alkylating agents, pan-DUB inhibitors combined with probe labelling 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.

Keywords: Deubiquitylating enzymes (DUBs), Mass Spectrometry, Proteomics, Chemical Biology, ubiquitin specific peptidase, isoforms

Received: 15 Jun 2019; Accepted: 09 Aug 2019.

Copyright: © 2019 Pinto-Fernández, Davis, Schofield, Scott, Zhang, Salah, Mathea, Charles, Damianou, Bond, Fischer and Kessler. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence:
Dr. Adán Pinto-Fernández, University of Oxford, Oxford, United Kingdom, adan.pintofernandez@ndm.ox.ac.uk
Prof. Benedikt M. Kessler, University of Oxford, Oxford, United Kingdom, benedikt.kessler@ndm.ox.ac.uk