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

Samuel Kasson,In-Kwon Kim,Nuwani Dharmapriya

doi:10.1371/journal.pone.0254022

Samuel Kasson, In-Kwon Kim + Show 1 more

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https://doi.org/10.1371/journal.pone.0254022

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Journal: PloS one	Publication Date: Jun 30, 2021
Citations: 7	License type: CC BY 4.0

Affiliation: University of Cincinnati

Abstract

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+. 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.

Highlights

ADP-ribosylation is a reversible post translational modification (PTM) that regulates many cellular signaling pathways, including DNA repair, DNA replication, gene expression, and cell death [1,2,3,4]
The ADP-ribosylation cycle is a tightly controlled process involving a variety of different enzymes classified as ADP-ribose writers that add monomeric or polymeric ADPribose units, readers that interact with ADP-ribose units to mediate cellular signaling, and erasers that cleave poly- or site-specific mono-ADP-ribosylations [5,6,7,8,9,10]
ADP-ribosylation has been proven as a key PTM that is responsible for a variety of cellular processes, the development of tools to quantitatively study the reversal of different types of ADP-ribosylations has been lagging

Summary

Introduction

ADP-ribosylation is a reversible post translational modification (PTM) that regulates many cellular signaling pathways, including DNA repair, DNA replication, gene expression, and cell death [1,2,3,4]. The ADP-ribosylation cycle is a tightly controlled process involving a variety of different enzymes classified as ADP-ribose writers that add monomeric or polymeric ADPribose units, readers that interact with ADP-ribose units to mediate cellular signaling, and erasers that cleave poly- or site-specific mono-ADP-ribosylations [5,6,7,8,9,10]. Upon DNA damage, PARP1 becomes enzymatically activated and incorporates polymeric ADP-ribose chains onto itself and target proteins, using NAD+ as a substrate, to signal DNA damage. The importance of PAR turnover is further highlighted by the embryonic lethal phenotype found in PARG–/–mice [17]

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