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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Autores principales: Samuel Kasson, Nuwani Dharmapriya, In-Kwon Kim
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Publicado: Public Library of Science (PLoS) 2021
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Acceso en línea:https://doaj.org/article/9340367bbbce459eb9ed7f31ae9848b4
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spelling oai:doaj.org-article:9340367bbbce459eb9ed7f31ae9848b42021-12-02T20:15:42ZSelective monitoring of the protein-free ADP-ribose released by ADP-ribosylation reversal enzymes.1932-620310.1371/journal.pone.0254022https://doaj.org/article/9340367bbbce459eb9ed7f31ae9848b42021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0254022https://doaj.org/toc/1932-6203ADP-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.Samuel KassonNuwani DharmapriyaIn-Kwon KimPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 6, p e0254022 (2021)
institution DOAJ
collection DOAJ
language EN
topic Medicine
R
Science
Q
spellingShingle Medicine
R
Science
Q
Samuel Kasson
Nuwani Dharmapriya
In-Kwon Kim
Selective monitoring of the protein-free ADP-ribose released by ADP-ribosylation reversal enzymes.
description 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.
format article
author Samuel Kasson
Nuwani Dharmapriya
In-Kwon Kim
author_facet Samuel Kasson
Nuwani Dharmapriya
In-Kwon Kim
author_sort Samuel Kasson
title Selective monitoring of the protein-free ADP-ribose released by ADP-ribosylation reversal enzymes.
title_short Selective monitoring of the protein-free ADP-ribose released by ADP-ribosylation reversal enzymes.
title_full Selective monitoring of the protein-free ADP-ribose released by ADP-ribosylation reversal enzymes.
title_fullStr Selective monitoring of the protein-free ADP-ribose released by ADP-ribosylation reversal enzymes.
title_full_unstemmed Selective monitoring of the protein-free ADP-ribose released by ADP-ribosylation reversal enzymes.
title_sort selective monitoring of the protein-free adp-ribose released by adp-ribosylation reversal enzymes.
publisher Public Library of Science (PLoS)
publishDate 2021
url https://doaj.org/article/9340367bbbce459eb9ed7f31ae9848b4
work_keys_str_mv AT samuelkasson selectivemonitoringoftheproteinfreeadpribosereleasedbyadpribosylationreversalenzymes
AT nuwanidharmapriya selectivemonitoringoftheproteinfreeadpribosereleasedbyadpribosylationreversalenzymes
AT inkwonkim selectivemonitoringoftheproteinfreeadpribosereleasedbyadpribosylationreversalenzymes
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