1. Academic Validation
  2. Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate

Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate

  • Cell Chem Biol. 2018 Dec 20;25(12):1562-1570.e19. doi: 10.1016/j.chembiol.2018.09.008.
Bryon S Drown 1 Tomohiro Shirai 1 Johannes Gregor Matthias Rack 2 Ivan Ahel 2 Paul J Hergenrother 3
Affiliations

Affiliations

  • 1 Department of Chemistry and Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 261 Roger Adams Lab Box 36-5, 600 S. Mathews Avenue, Urbana, IL 61801, USA.
  • 2 Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
  • 3 Department of Chemistry and Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 261 Roger Adams Lab Box 36-5, 600 S. Mathews Avenue, Urbana, IL 61801, USA. Electronic address: hergenro@illinois.edu.
Abstract

The post-translational modification (PTM) and signaling molecule poly(ADP-ribose) (PAR) has an impact on diverse biological processes. This PTM is regulated by a series of ADP-ribosyl glycohydrolases (PARG Enzymes) that cleave Polymers and/or liberate monomers from their protein targets. Existing methods for monitoring these hydrolases rely on detection of the natural substrate, PAR, commonly achieved via radioisotopic labeling. Here we disclose a general substrate for monitoring PARG activity, TFMU-ADPr, which directly reports on total PAR hydrolase activity via release of a fluorophore; this substrate has excellent reactivity, generality (processed by the major PARG Enzymes), stability, and usability. A second substrate, TFMU-IDPr, selectively reports on PARG activity only from the Enzyme ARH3. Use of these probes in whole-cell lysate experiments has revealed a mechanism by which ARH3 is inhibited by cholera toxin. TFMU-ADPr and TFMU-IDPr are versatile tools for assessing small-molecule inhibitors in vitro and probing the regulation of ADP-ribosyl catabolic Enzymes.

Keywords

ARH3; PARG; cholera toxin; enzyme assay; fluorescent probe; poly(ADP-ribose).

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