Abstract

Activity-based probes (ABPs) are widely used to monitor the activity of enzyme families in biological systems. Inferring enzyme activity from probe reactivity requires that the probe reacts with the enzyme at its active site; however, probe-labeling sites are rarely verified. Here we present an enhanced chemoproteomic approach to evaluate the activity and probe reactivity of deubiquitinase enzymes, using bioorthogonally tagged ABPs and a sequential on-bead digestion protocol to enhance the identification of probe-labeling sites. We confirm probe labeling of deubiquitinase catalytic Cys residues and reveal unexpected labeling of deubiquitinases on non-catalytic Cys residues and of non-deubiquitinase proteins. In doing so, we identify ZUFSP (ZUP1) as a previously unannotated deubiquitinase with high selectivity toward cleaving K63-linked chains. ZUFSP interacts with and modulates ubiquitination of the replication protein A (RPA) complex. Our reactive-site-centric chemoproteomics method is broadly applicable for identifying the reaction sites of covalent molecules, which may expand our understanding of enzymatic mechanisms.

Details

Title
Reactive-site-centric chemoproteomics identifies a distinct class of deubiquitinase enzymes
Author
Hewings, David S 1   VIAFID ORCID Logo  ; Heideker, Johanna 2 ; Ma, Taylur P 3 ; AhYoung, Andrew P 4 ; Farid El Oualid 5   VIAFID ORCID Logo  ; Amore, Alessia 5 ; Costakes, Gregory T 6 ; Kirchhofer, Daniel 4   VIAFID ORCID Logo  ; Brasher, Bradley 6 ; Pillow, Thomas 7   VIAFID ORCID Logo  ; Popovych, Nataliya 4 ; Maurer, Till 8 ; Schwerdtfeger, Carsten 6 ; Forrest, William F 9 ; Yu, Kebing 3 ; Flygare, John 10 ; Bogyo, Matthew 11 ; Wertz, Ingrid E 2 

1  Discovery Oncology, Genentech, South San Francisco, California, USA; Early Discovery Biochemistry, Genentech, South San Francisco, California, USA; Discovery Chemistry, Genentech, South San Francisco, California, USA; Department of Pathology, Stanford University School of Medicine, Stanford, California, USA 
2  Discovery Oncology, Genentech, South San Francisco, California, USA; Early Discovery Biochemistry, Genentech, South San Francisco, California, USA 
3  Microchemistry, Proteomics and Lipidomics, Genentech, South San Francisco, CA, USA 
4  Early Discovery Biochemistry, Genentech, South San Francisco, California, USA 
5  UbiQ Bio BV, Amsterdam, The Netherlands 
6  Boston Biochem Inc., Cambridge, Massachussetts, USA 
7  Discovery Chemistry, Genentech, South San Francisco, California, USA 
8  Structural Biology, Genentech, South San Francisco, California, USA 
9  Bioinformatics, Genentech, South San Francisco, California, USA 
10  Discovery Chemistry, Genentech, South San Francisco, California, USA; Merck, South San Francisco, California, USA 
11  Department of Pathology, Stanford University School of Medicine, Stanford, California, USA 
Pages
1-17
Publication year
2018
Publication date
Mar 2018
Publisher
Nature Publishing Group
e-ISSN
20411723
Source type
Scholarly Journal
Language of publication
English
DOI
https://doi.org/10.1038/s41467-018-03511-6
ProQuest document ID
2016523602
Copyright
© 2018. This work is published under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.