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

Bryon S. Drown, Tomohiro Shirai, Johannes Gregor Matthias Rack, Ivan Ahel, Paul J. Hergenrother

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)1562-1570.e19
JournalCell chemical biology
Volume25
Issue number12
DOIs
StatePublished - Dec 20 2018

Fingerprint

Glycoside Hydrolases
Adenosine Diphosphate
Hydrolases
Post Translational Protein Processing
Monitoring
Substrates
Enzymes
Poly Adenosine Diphosphate Ribose
Biological Phenomena
Enzyme Stability
Cholera Toxin
Molecules
Fluorophores
Polymers
Labeling
Monomers
Proteins
Experiments

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmacology
  • Drug Discovery
  • Clinical Biochemistry

Cite this

Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate. / Drown, Bryon S.; Shirai, Tomohiro; Rack, Johannes Gregor Matthias; Ahel, Ivan; Hergenrother, Paul J.

In: Cell chemical biology, Vol. 25, No. 12, 20.12.2018, p. 1562-1570.e19.

Research output: Contribution to journalArticle

Drown, Bryon S. ; Shirai, Tomohiro ; Rack, Johannes Gregor Matthias ; Ahel, Ivan ; Hergenrother, Paul J. / Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate. In: Cell chemical biology. 2018 ; Vol. 25, No. 12. pp. 1562-1570.e19.
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