Structural and Functional Characterization of Ubiquitin Variant Inhibitors of USP15

Joan Teyra; Alex U Singer; Frank W Schmitges; Patrick Jaynes; Sarah Kit Leng Lui; Maria J Polyak; Nassima Fodil; Jonathan R Krieger; Jiefei Tong; Carsten Schwerdtfeger; Bradley B Brasher; Derek F J Ceccarelli; Jason Moffat; Frank Sicheri; Michael F Moran; Philippe Gros; Pieter J A Eichhorn; Martin Lenter; Guido Boehmelt; Sachdev S Sidhu

doi:10.1016/j.str.2019.01.002

Structural and Functional Characterization of Ubiquitin Variant Inhibitors of USP15

Structure. 2019 Apr 2;27(4):590-605.e5. doi: 10.1016/j.str.2019.01.002. Epub 2019 Jan 31.

Authors

Joan Teyra¹, Alex U Singer¹, Frank W Schmitges¹, Patrick Jaynes², Sarah Kit Leng Lui², Maria J Polyak³, Nassima Fodil³, Jonathan R Krieger⁴, Jiefei Tong⁵, Carsten Schwerdtfeger⁶, Bradley B Brasher⁶, Derek F J Ceccarelli⁷, Jason Moffat⁸, Frank Sicheri⁹, Michael F Moran¹⁰, Philippe Gros¹¹, Pieter J A Eichhorn¹², Martin Lenter¹³, Guido Boehmelt¹⁴, Sachdev S Sidhu¹⁵

Affiliations

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.

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

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Catalytic Domain
Cloning, Molecular
Crystallography, X-Ray
Escherichia coli / genetics
Escherichia coli / metabolism
Gene Expression
Genetic Vectors / chemistry
Genetic Vectors / metabolism
HEK293 Cells
Humans
Models, Molecular
Protein Binding
Protein Conformation, alpha-Helical
Protein Conformation, beta-Strand
Protein Interaction Domains and Motifs
Protein Multimerization
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Recombinant Proteins / metabolism
Sequence Alignment
Sequence Homology, Amino Acid
Substrate Specificity
Transcription Factors / chemistry*
Transcription Factors / genetics
Transcription Factors / metabolism
Transforming Growth Factor beta1 / chemistry*
Transforming Growth Factor beta1 / genetics
Transforming Growth Factor beta1 / metabolism
Tripartite Motif Proteins / chemistry*
Tripartite Motif Proteins / genetics
Tripartite Motif Proteins / metabolism
Ubiquitin / chemistry*
Ubiquitin / genetics
Ubiquitin / metabolism
Ubiquitin-Protein Ligases / chemistry*
Ubiquitin-Protein Ligases / genetics
Ubiquitin-Protein Ligases / metabolism
Ubiquitin-Specific Proteases / antagonists & inhibitors
Ubiquitin-Specific Proteases / chemistry*
Ubiquitin-Specific Proteases / genetics
Ubiquitin-Specific Proteases / metabolism
Ubiquitination

Substances

Recombinant Proteins
TGFB1 protein, human
Transcription Factors
Transforming Growth Factor beta1
Tripartite Motif Proteins
Ubiquitin
SMURF2 protein, human
TRIM25 protein, human
Ubiquitin-Protein Ligases
USP15 protein, human
Ubiquitin-Specific Proteases