UbFluor: A Fluorescent Thioester to Monitor HECT E3 Ligase Catalysis. Issue 1 (2nd March 2017)

• Record Type: Journal Article
• Authors: Krist, David T.; Foote, Peter K.; Statsyuk, Alexander V.
• Is Part Of: Current protocols in chemical biology. Volume 9:Issue 1(2017)
• Page Start: 11
• Page End: 37
• Publication Date: 2017-03-02
• Subjects: fluorescence polarization -- HECT ligase -- thioester -- ubiquitin; Biochemistry -- Laboratory manuals; Chemical Phenomena; Biochemistry; Periodicals; Laboratory manuals; 572.078
• DOI: 10.1002/cpch.17
• Languages: English
• ISSNs: 2160-4762

Abstract

Abstract : HECT E3 ubiquitin ligases (∼28 are known) are associated with many phenotypes in eukaryotes and are important drug targets. However, assays used to screen for small molecule inhibitors of HECT E3s are complex and require ATP, Ub, E1, E2, and HECT E3 enzymes, producing three covalent thioester enzyme intermediates E1∼Ub, E2∼Ub, and HECT E3∼Ub (where ∼ indicates a thioester bond), and mixtures of polyubiquitin chains. To reduce the complexity of the assay, we developed a novel class of fluorescent probes, UbFluor, that act as mechanistically relevant pseudosubstrates of HECT E3s. These probes undergo a direct transthiolation reaction with the catalytic cysteine of HECT E3s, producing the catalytically active HECT E3∼Ub thioester accompanied by fluorophore release. Thus, a fluorescence polarization assay can continuously monitor UbFluor consumption by HECT E3s, and changes in UbFluor consumption rendered by biochemical point mutations or small molecule modulation of HECT E3 activity. © 2017 by John Wiley & Sons, Inc.