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Structure. 2019 Jan 17. pii: S0969-2126(19)30002-4. doi: 10.1016/j.str.2019.01.002. [Epub ahead of print]

Structural and Functional Characterization of Ubiquitin Variant Inhibitors of USP15.

Author information

1
The Donnelly Centre, University of Toronto, Toronto, ON M5S 3E1, Canada.
2
Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore.
3
Department of Biochemistry, McGill University, Montreal, QC, Canada; Corbin Therapeutics, Montreal, QC, Canada.
4
SPARC BioCentre, Hospital for Sick Children, Toronto, ON M5G 0A4, Canada.
5
Cell Biology Program, The Hospital for Sick Children, 686 Bay Street, Toronto, ON M5G 0A4, Canada.
6
Boston Biochem, a Bio-Techne Brand, 840 Memorial Drive, Cambridge, MA 02139, USA.
7
Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada.
8
The Donnelly Centre, University of Toronto, Toronto, ON M5S 3E1, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada.
9
Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada.
10
SPARC BioCentre, Hospital for Sick Children, Toronto, ON M5G 0A4, Canada; Cell Biology Program, The Hospital for Sick Children, 686 Bay Street, Toronto, ON M5G 0A4, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada.
11
Department of Biochemistry, McGill University, Montreal, QC, Canada.
12
Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore; Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
13
Boehringer Ingelheim Pharma GmbH & Co KG, Biberach, Germany.
14
Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria.
15
The Donnelly Centre, University of Toronto, Toronto, ON M5S 3E1, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address: sachdev.sidhu@utoronto.ca.

Abstract

The multi-domain deubiquitinase USP15 regulates diverse eukaryotic processes and has been implicated in numerous diseases. We developed ubiquitin variants (UbVs) that targeted either the catalytic domain or each of three adaptor domains in USP15, including the N-terminal DUSP domain. We also designed a linear dimer (diUbV), which targeted the DUSP and catalytic domains, and exhibited enhanced specificity and more potent inhibition of catalytic activity than either UbV alone. In cells, the UbVs inhibited the deubiquitination of two USP15 substrates, SMURF2 and TRIM25, and the diUbV inhibited the effects of USP15 on the transforming growth factor β pathway. Structural analyses revealed that three distinct UbVs bound to the catalytic domain and locked the active site in a closed, inactive conformation, and one UbV formed an unusual strand-swapped dimer and bound two DUSP domains simultaneously. These inhibitors will enable the study of USP15 function in oncology, neurology, immunology, and inflammation.

KEYWORDS:

DUSP; TGF-β pathway; USP15; USP4; UbV; catalytic domain; deubiquitinase; phage display; ubiquitin; ubiquitin-like

PMID:
30713027
DOI:
10.1016/j.str.2019.01.002

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