alexa Broad Utility of an Affinity-enrichment Strategy for Unanchored Polyubiquitin Chains
ISSN: 0974-276X

Journal of Proteomics & Bioinformatics
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Research Article

Broad Utility of an Affinity-enrichment Strategy for Unanchored Polyubiquitin Chains

Jo Strachan1, Barry Shaw1, Varun Gopala Krishna1, Daniel Scott1, Juan C del Pozo2, Kristine Hill3, Madhav Gautam4, Dorota Skowyra4, Andrew D. Jacobson5, Chang-Wei Liu5, Neil J Oldham6 and Rob Layfield1*

1School of Life Sciences, University of Nottingham, Nottingham, UK

2Centro de Biotecnología y Genómica de Plantas INIA-UPM, Madrid, Spain

3Centre for Plant Integrative Biology, University of Nottingham, Nottingham, UK

4Edward A Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri, USA

5Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado, USA

6School of Chemistry, University of Nottingham, Nottingham, UK

*Corresponding Author:
Rob Layfield
School of Life Sciences
University of Nottingham
Nottingham NG7 2UH, United Kingdom
Tel: +44-115-8230107
E-mail: [email protected]

Received Date: September 18, 2013; Accepted Date: November 01, 2013; Published Date: November 04, 2013

Citation: Strachan J, Shaw B, Krishna VG, Scott D, del Pozo JC, et al. (2013) Broad Utility of an Affinity-enrichment Strategy for Unanchored Polyubiquitin Chains. J Proteomics Bioinform S7:001. doi:10.4172/jpb.S7-001

Copyright: © 2013 Strachan J, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

 

Abstract

Protein ubiquitination is a common post-translational modification where selected targets are covalently modified by the ubiquitin protein, often in the form of isopeptide-linked polyubiquitin chains. More recently, unanchored (i.e. non-substrate-linked) polyubiquitin chains have also been described and implicated in a range of biological processes. The development of Tandem-repeated Ubiquitin-Binding Entities (TUBEs), engineered repeats of ubiquitin-binding domains that interact non-covalently with polyubiquitin, has allowed strategies for the affinity-enrichment of ubiquitinmodified proteins to be established, in some cases with linkage specificity. Here, we demonstrate the utility of a Free Ubiquitin-Binding Entity (FUBE), based on an ubiquitin-binding domain with high specificity for the free C-terminus of ubiquitin (the Znf-UBP domain of human USP5). In contrast to TUBEs which do not distinguish conjugated or free polyubiquitin, the FUBE exclusively recognises ubiquitin in its unconjugated form, including endogenous unanchored polyubiquitin chains. Affinity-enrichments using the FUBE demonstrate that unanchored polyubiquitin chains are present in different mammalian cell lines and accumulate when the 26S proteasome is pharmacologically inhibited, being retained on the proteasome. The high conservation of the ubiquitin sequence permits the FUBE to also be applied to the purification of endogenous unanchored polyubiquitin chains from species as diverse as Arabidopsis thaliana and Saccharomyces cerevisiae. The development and refinement of an affinity-enrichment strategy for unanchored polyubiquitin chains opens the way for more complete investigations into their biological significance.

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