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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2021
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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) |
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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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1718374540804882432 |