title: Subunit-Specific Labeling of Ubiquitin Chains by Using Sortase: Insights into the Selectivity of Deubiquitinases.
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.
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA.
authors.forename: Sean O
authors.initials: SO
authors.lastname: Crowe
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA.
authors.forename: Grace H
authors.initials: GH
authors.lastname: Pham
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA.
authors.forename: Jacob C
authors.initials: JC
authors.lastname: Ziegler
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA.
authors.forename: Kirandeep K
authors.initials: KK
authors.lastname: Deol
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA.
authors.forename: Robert G
authors.initials: RG
authors.lastname: Guenette
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA.|Department of Cell and Regenerative Biology, University of Wisconsin-Madison School of Medicine and Public Health, Madison, WI, 53706, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Ge
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA. strieter@chem.wisc.edu.
authors.forename: Eric R
authors.initials: ER
authors.lastname: Strieter
mesh_terms: D019881:Aminoacyltransferases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D055162:Biocatalysis; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D000072017:Deubiquitinating Enzymes / Q000378:metabolism*; D006801:Humans; D008968:Molecular Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D021122:Protein Subunits; D025801:Ubiquitin; D019881:Aminoacyltransferases; C120542:sortase A; D000072017:Deubiquitinating Enzymes; D003546:Cysteine Endopeptidases
keywords: N-terminal modification; deubiquitinases; fluorophore labeling; sortase; ubiquitin chains