Subunit‐Specific Labeling of Ubiquitin Chains by Using Sortase: Insights into the Selectivity of Deubiquitinases. (4th July 2016)

• Record Type: Journal Article
• Authors: Crowe, Sean O.; Pham, Grace H.; Ziegler, Jacob C.; Deol, Kirandeep K.; Guenette, Robert G.; Ge, Ying; Strieter, Eric R.
• Is Part Of: Chembiochem. Volume 17:Number 16(2016)
• Page Start: 1525
• Page End: 1531
• Publication Date: 2016-07-04
• Subjects: deubiquitinases -- fluorophore labeling -- N-terminal modification -- sortase -- ubiquitin chains; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.201600276
• Languages: English
• ISSNs: 1439-4227

Abstract

Abstract: Information embedded in different ubiquitin chains is transduced by proteins with ubiquitin‐binding domains (UBDs) and erased by a set of hydrolytic enzymes referred to as deubiquitinases (DUBs). Understanding the selectivity of UBDs and DUBs is necessary for decoding the functions of different ubiquitin chains. Critical to these efforts is the access to chemically defined ubiquitin chains bearing site‐specific fluorescent labels. One approach toward constructing such molecules involves peptide ligation by sortase (SrtA), a bacterial transpeptidase responsible for covalently attaching cell surface proteins to the cell wall. Here, we demonstrate the utility of SrtA in modifying individual subunits of ubiquitin chains. Using ubiquitin derivatives in which an N‐terminal glycine is unveiled after protease‐mediated digestion, we synthesized ubiquitin dimers, trimers, and tetramers with different isopeptide linkages. SrtA was then used in combination with fluorescent depsipeptide substrates to effect the modification of each subunit in a chain. By constructing branched ubiquitin chains with individual subunits tagged with a fluorophore, we provide evidence that the ubiquitin‐specific protease USP15 prefers ubiquitin trimers but has little preference for a particular isopeptide linkage. Our results emphasize the importance of subunit‐specific labeling of ubiquitin chains when studying how DUBs process these chains. Abstract : Subunit sortagging : Sortagging is used to fluorescently label individual subunits of ubiquitin chains. This method enables the biochemical characterization of deubiquitinases by examining the selectivity toward specific isopeptide linkages and chain topologies. In this work, we demonstrate that the ubiquitin‐specific protease USP15 prefers branched over linear ubiquitin chains.