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

David S. Hewings, Johanna Heideker, Taylur P. Ma, Andrew P. AhYoung, Farid El Oualid, Alessia Amore, Gregory T. Costakes, Daniel Kirchhofer, Bradley Brasher, Thomas Pillow, Nataliya Popovych, Till Maurer, Carsten Schwerdtfeger, William F. Forrest, Kebing Yu, John Flygare, Matthew Bogyo and Ingrid E. Wertz ()
Additional contact information
David S. Hewings: Genentech
Johanna Heideker: Genentech
Taylur P. Ma: Genentech
Andrew P. AhYoung: Genentech
Farid El Oualid: UbiQ Bio BV
Alessia Amore: UbiQ Bio BV
Gregory T. Costakes: Boston Biochem Inc.
Daniel Kirchhofer: Genentech
Bradley Brasher: Boston Biochem Inc.
Thomas Pillow: Genentech
Nataliya Popovych: Genentech
Till Maurer: Genentech
Carsten Schwerdtfeger: Boston Biochem Inc.
William F. Forrest: Genentech
Kebing Yu: Genentech
John Flygare: Genentech
Matthew Bogyo: Stanford University School of Medicine
Ingrid E. Wertz: Genentech

Nature Communications, 2018, vol. 9, issue 1, 1-17

Abstract: 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.

Date: 2018
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DOI: 10.1038/s41467-018-03511-6

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