Ubiquitin-C-terminal hydrolases cleave isopeptide and peptide-linked ubiquitin from structured proteins but do not edit ubiquitin homopolymers

Bett, J.S. et al. (2015) Ubiquitin-C-terminal hydrolases cleave isopeptide and peptide-linked ubiquitin from structured proteins but do not edit ubiquitin homopolymers. Biochemical Journal, 466(3), pp. 489-498. (doi: 10.1042/BJ20141349) (PMID:25489924) (PMCID:PMC4353193)

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Modification of proteins with ubiquitin (Ub) occurs through a variety of topologically distinct Ub linkages, including Ube2W-mediated monoubiquitylation of N-terminal alpha amines to generate peptide-linked linear mono-Ub fusions. Protein ubiquitylation can be reversed by the action of deubiquitylating enzymes (DUBs), many of which show striking preference for particular Ub linkage types. Here, we have screened for DUBs that preferentially cleave N-terminal Ub from protein substrates but do not act on ubiquitin homopolymers. We show that members of the Ub C-terminal hydrolase (UCH) family of DUBs demonstrate this preference for N-terminal deubiquitylating activity as they are capable of cleaving N-terminal Ub from SUMO2 and Ube2W, while displaying no activity against any of the 8 Ub linkage types. Surprisingly, this ability to cleave Ub from SUMO2 was 100 times more efficient for UCH-L3 when we deleted the unstructured N-terminus of SUMO2, demonstrating that UCH enzymes can cleave Ub from structured proteins. However, UCH-L3 could also cleave chemically synthesised isopeptide-linked Ub from Lysine 11 (K11) of SUMO2 with similar efficiency, demonstrating that UCH DUB activity is not limited to peptide-linked Ub. These findings advance our understanding of the specificity of the UCH family of DUBs, which are strongly implicated in cancer and neurodegeneration but whose substrate-preference has remained unclear. In addition, our findings suggest that the reversal of Ube2W-mediated N-terminal ubiquitylation may be one physiological role of UCH DUBs in vivo.

Item Type:Articles
Additional Information:Work in the RTH laboratory was funded by Cancer Research UK [programme grant number C434/A13067] and a Wellcome Trust Senior Investigator Award [grant number 098391/Z/12/Z]. J.W.C. is supported by the Medical Research Council [grant numbers U105181009 and UD99999908].
Glasgow Author(s) Enlighten ID:Kurz, Dr Thimo and Bett, Dr John
Authors: Bett, J.S., Ritorto, M.S., Ewan, R., Jaffray, E., Virdee, S., Chin, J., Knebel, A., Kurz, T., Trost, M., Tatham, M., and Hay, R.
College/School:College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
Journal Name:Biochemical Journal
Publisher:Portland Press Ltd.
ISSN (Online):1470-8728
Published Online:09 December 2014
Copyright Holders:Copyright © 2015 The Authors
First Published:First published in Biochemical Journal 466(3): 489-498
Publisher Policy:Reproduced under a Creative Commons License

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