Comprehensive Landscape of Active Deubiquitinating Enzymes Profiled by Advanced Chemoproteomics

Frontiers in Chemistry

Abigail B. Schofield¹  Sebastian Mathea³  Ping Zhang⁴  Gareth Bond⁴  Eidarus Salah⁵  Simon Davis⁶  Philip D. Charles⁶  Andreas Damianou⁶  Roman Fischer⁶  Hannah C. Scott⁶  Benedikt M. Kessler⁶  Adán Pinto-Fernández⁶ 

[1] Christ Church, University of Oxford, Oxford, United Kingdom;Department of Chemistry, University of Oxford, Oxford, United Kingdom;Institute of Pharmaceutical Chemistry, Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Frankfurt, Germany;Ludwig Institute for Cancer Research, University of Oxford, Oxford, United Kingdom;Structural Genomics Consortium (United Kingdom), Oxford, United Kingdom;Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom;University of Oxford, Oxford, United Kingdom;

关键词: deubiquitylating enzymes;    mass spectrometry;    proteomics;    chemical biology;    ubiquitin specific proteases;    isoforms;   

DOI  :  10.3389/fchem.2019.00592

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown