Chemoenzymatic Synthesis of Bifunctional Polyubiquitin Substrates for Monitoring Ubiquitin Chain Remodeling. Issue 11 (24th June 2014)

• Record Type: Journal Article
• Authors: Trang, Vivian H.; Rodgers, Margaret L.; Boyle, Kevin J.; Hoskins, Aaron A.; Strieter, Eric R.
• Is Part Of: Chembiochem. Volume 15:Issue 11(2014)
• Page Start: 1563
• Page End: 1568
• Publication Date: 2014-06-24
• Subjects: Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.201402059
• Languages: English
• ISSNs: 1439-4227

Abstract

<abstract abstract-type="main" xml:lang="en"> <title>Abstract</title> <p>Covalent attachment of ubiquitin to target proteins is one of the most pervasive post‐translational modifications in eukaryotes. Target proteins are often modified with polymeric ubiquitin chains of defined lengths and linkages that may further undergo dynamic changes in composition in response to cellular signals. Biochemical characterization of the enzymes responsible for building and destroying ubiquitin chains is often thwarted by the lack of methods for preparation of the appropriate substrates containing probes for biochemical or biophysical studies. We have discovered that a yeast ubiquitin C‐terminal hydrolase (Yuh1) also catalyzes transamidation reactions that can be exploited to prepare site‐specifically modified polyubiquitin chains produced by thiol‐ene chemistry. We have used this chemoenzymatic approach to prepare dual‐functionalized ubiquitin chains containing fluorophore and biotin modifications. These dual‐functionalized ubiquitin chains enabled the first real‐time assay of ubiquitin chain disassembly by a human deubiquitinase (DUB) enzyme by single molecule fluorescence microscopy. In summary, this work provides a powerful new tool for elucidating the mechanisms of DUBs and other ubiquitin processing enzymes.</p> </abstract>