Reactive-site-centric chemoproteomics identifies a distinct class of deubiquitinase enzymes

Nat Commun. 2018 Mar 21;9(1):1162. doi: 10.1038/s41467-018-03511-6.

David S Hewings ¹ ² ³ ⁴, Johanna Heideker ¹ ², Taylur P Ma ⁵, Andrew P AhYoung ², Farid El Oualid ⁶, Alessia Amore ⁶, Gregory T Costakes ⁷, Daniel Kirchhofer ², Bradley Brasher ⁷, Thomas Pillow ³, Nataliya Popovych ², Till Maurer ⁸, Carsten Schwerdtfeger ⁷, William F Forrest ⁹, Kebing Yu ⁵, John Flygare ³ ¹⁰, Matthew Bogyo ⁴, Ingrid E Wertz ¹¹ ¹²

Affiliations

1 Discovery Oncology, Genentech, South San Francisco, California, 94080, USA.
2 Early Discovery Biochemistry, Genentech, South San Francisco, California, 94080, USA.
3 Discovery Chemistry, Genentech, South San Francisco, California, 94080, USA.
4 Department of Pathology, Stanford University School of Medicine, Stanford, California, 94305, USA.
5 Microchemistry, Proteomics and Lipidomics, Genentech, South San Francisco, CA, 94080, USA.
6 UbiQ Bio BV, Science Park 408, 1098 XH, Amsterdam, The Netherlands.
7 Boston Biochem Inc., 840 Memorial Drive, Cambridge, Massachussetts, 02139, USA.
8 Structural Biology, Genentech, South San Francisco, California, 94080, USA.
9 Bioinformatics, Genentech, South San Francisco, California, 94080, USA.
10 Merck, 630 Gateway Boulevard, South San Francisco, California, 94080, USA.
11 Discovery Oncology, Genentech, South San Francisco, California, 94080, USA. wertz.ingrid@gene.com.
12 Early Discovery Biochemistry, Genentech, South San Francisco, California, 94080, USA. wertz.ingrid@gene.com.

PMID: 29563501 DOI: 10.1038/s41467-018-03511-6

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.

Name
Email *

	Sorry, but the email address you supplied was invalid.