Journal of Proteomics & Bioinformatics

Journal of Proteomics & Bioinformatics
Open Access

ISSN: 0974-276X

+44 1223 790975


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

Jo Strachan, Barry Shaw, Varun Gopala Krishna, Daniel Scott, Juan C del Pozo, Kristine Hill, Madhav Gautam, Dorota Skowyra, Andrew D. Jacobson, Chang-Wei Liu, Neil J Oldham and Rob Layfield

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.