Selective monitoring of the protein-free ADP-ribose released by ADP-ribosylation reversal enzymes

ADP-ribosylation is a key post-translational modification that regulates a wide variety of cellular stress responses. The ADP-ribosylation cycle is maintained by writers and erasers. For example, poly(ADP-ribosyl)ation cycles consist of two predominant enzymes, poly(ADP-ribose) polymerases (PARPs) a...

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Bibliographic Details
Published in:PloS one 2021-06, Vol.16 (6), p.e0254022-e0254022
Main Authors: Kasson, Samuel, Dharmapriya, Nuwani, Kim, In-Kwon
Format: Article
Language:English
Subjects:
Adenosine diphosphate
ADP-ribosylation
AMP
Analysis
Apoptosis
Assaying
Biology and Life Sciences
Cellular stress response
Chemistry
Cloning
DNA damage
Enzymes
Gene expression
Medicine and Health Sciences
Metabolism
NAD
Physical sciences
Plasmids
Poly(ADP-ribose)
Poly(ADP-ribose) glycohydrolase
Poly(ADP-ribose) polymerase
Post-translation
Post-translational modification
Protein binding
Proteins
Research and Analysis Methods
Ribose
Ribosylation
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Summary:ADP-ribosylation is a key post-translational modification that regulates a wide variety of cellular stress responses. The ADP-ribosylation cycle is maintained by writers and erasers. For example, poly(ADP-ribosyl)ation cycles consist of two predominant enzymes, poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribose) glycohydrolase (PARG). However, historically, mechanisms of erasers of ADP-ribosylations have been understudied, primarily due to the lack of quantitative tools to selectively monitor specific activities of different ADP-ribosylation reversal enzymes. Here, we developed a new NUDT5-coupled AMP-Glo (NCAG) assay to specifically monitor the protein-free ADP-ribose released by ADP-ribosylation reversal enzymes. We found that NUDT5 selectively cleaves protein-free ADP-ribose, but not protein-bound poly- and mono-ADP-ribosylations, protein-free poly(ADP-ribose) chains, or NAD.sup.+ . As a proof-of-concept, we successfully measured the kinetic parameters for the exo-glycohydrolase activity of PARG, which releases monomeric ADP-ribose, and monitored activities of site-specific mono-ADP-ribosyl-acceptor hydrolases, such as ARH3 and TARG1. This NCAG assay can be used as a general platform to study the mechanisms of diverse ADP-ribosylation reversal enzymes that release protein-free ADP-ribose as a product. Furthermore, this assay provides a useful tool to identify small-molecule probes targeting ADP-ribosylation metabolism and to quantify ADP-ribose concentrations in cells.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0254022