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Science 339 (6119): 590-595

Copyright © 2013 by the American Association for the Advancement of Science

A Strategy for Modulation of Enzymes in the Ubiquitin System

Andreas Ernst,1 George Avvakumov,2 Jiefei Tong,3 Yihui Fan,4 Yanling Zhao,4 Philipp Alberts,3 Avinash Persaud,3,5 John R. Walker,2 Ana-Mirela Neculai,1 Dante Neculai,2 Andrew Vorobyov,1 Pankaj Garg,1 Linda Beatty,1 Pak-Kei Chan,6 Yu-Chi Juang,7 Marie-Claude Landry,7 Christina Yeh,7,8 Elton Zeqiraj,7 Konstantina Karamboulas,1 Abdellah Allali-Hassani,2 Masoud Vedadi,2 Mike Tyers,6,7 Jason Moffat,1,8,9,10 Frank Sicheri,7,8 Laurence Pelletier,7,8 Daniel Durocher,7,8 Brian Raught,10 Daniela Rotin,3,5 Jianhua Yang,4 Michael F. Moran,3,8,9 Sirano Dhe-Paganon,2,11 Sachdev S. Sidhu1,8,9,10,*

Abstract: The ubiquitin system regulates virtually all aspects of cellular function. We report a method to target the myriad enzymes that govern ubiquitination of protein substrates. We used massively diverse combinatorial libraries of ubiquitin variants to develop inhibitors of four deubiquitinases (DUBs) and analyzed the DUB-inhibitor complexes with crystallography. We extended the selection strategy to the ubiquitin conjugating (E2) and ubiquitin ligase (E3) enzymes and found that ubiquitin variants can also enhance enzyme activity. Last, we showed that ubiquitin variants can bind selectively to ubiquitin-binding domains. Ubiquitin variants exhibit selective function in cells and thus enable orthogonal modulation of specific enzymatic steps in the ubiquitin system.

1 Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, Ontario M5S 3E1, Canada.
2 Structural Genomics Consortium, MaRS Centre, 101 College Street, Suite 700, Toronto, Ontario M5G 1L7, Canada.
3 Hospital for Sick Children, 101 College Street, Toronto, Ontario M5G 1L7, Canada.
4 Texas Children's Cancer Center, Department of Pediatrics, Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.
5 Biochemistry Department, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
6 Institut de Recherche en Immunologie et Cancérologie, Université de Montréal, Montreal, Quebec H3C 3J7, Canada.
7 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada.
8 Department of Molecular Genetics, University of Toronto, 160 College Street, Toronto, Ontario M5S 3E1, Canada.
9 Banting and Best Department of Medical Research, University of Toronto, 160 College Street, Toronto, Ontario M5S 3E1, Canada.
10 Ontario Cancer Institute and McLaughlin Centre for Molecular Medicine, University of Toronto, 101 College Street, Toronto, Ontario M5G 1L7, Canada.
11 Department of Physiology, University of Toronto, 101 College Street, Toronto, Ontario M5G 1L7, Canada.

* To whom correspondence should be addressed. E-mail: sachdev.sidhu{at}utoronto.ca

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