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MacroGreen, a simple tool for detection of ADP-ribosylated proteins

Enzymes in the PARP family partake in the regulation of vital cellular signaling pathways by ADP-ribosylating their targets. The roles of these signaling pathways in disease development and the de-regulation of several PARP enzymes in cancer cells have motivated the pursuit of PARP inhibitors for th...

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Autores principales: García-Saura, Antonio Ginés, Herzog, Laura K., Dantuma, Nico P., Schüler, Herwig
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8319303/
https://www.ncbi.nlm.nih.gov/pubmed/34321589
http://dx.doi.org/10.1038/s42003-021-02439-w
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author García-Saura, Antonio Ginés
Herzog, Laura K.
Dantuma, Nico P.
Schüler, Herwig
author_facet García-Saura, Antonio Ginés
Herzog, Laura K.
Dantuma, Nico P.
Schüler, Herwig
author_sort García-Saura, Antonio Ginés
collection PubMed
description Enzymes in the PARP family partake in the regulation of vital cellular signaling pathways by ADP-ribosylating their targets. The roles of these signaling pathways in disease development and the de-regulation of several PARP enzymes in cancer cells have motivated the pursuit of PARP inhibitors for therapeutic applications. In this rapidly expanding research area, availability of simple research tools will help assess the functions of ADP-ribosylation in a wider range of contexts. Here, we generated a mutant Af1521 macrodomain fused to green fluorescent protein (GFP) to generate a high-affinity ADP-ribosyl binding reagent. The resulting tool – which we call MacroGreen – is easily produced by expression in Escherichia coli, and can detect both mono-and poly-ADP-ribosylation of diverse proteins in vitro. Staining with MacroGreen allows detection of ADP-ribosylation at sites of DNA damage by fluorescence microscopy. MacroGreen can also be used to quantify modification of target proteins in overlay assays, and to screen for PARP inhibitors in high-throughput format with excellent assay statistics. We expect that this broadly applicable tool will facilitate ADP-ribosylation related discoveries, including by laboratories that do not specialize in this field.
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spelling pubmed-83193032021-08-03 MacroGreen, a simple tool for detection of ADP-ribosylated proteins García-Saura, Antonio Ginés Herzog, Laura K. Dantuma, Nico P. Schüler, Herwig Commun Biol Comment Enzymes in the PARP family partake in the regulation of vital cellular signaling pathways by ADP-ribosylating their targets. The roles of these signaling pathways in disease development and the de-regulation of several PARP enzymes in cancer cells have motivated the pursuit of PARP inhibitors for therapeutic applications. In this rapidly expanding research area, availability of simple research tools will help assess the functions of ADP-ribosylation in a wider range of contexts. Here, we generated a mutant Af1521 macrodomain fused to green fluorescent protein (GFP) to generate a high-affinity ADP-ribosyl binding reagent. The resulting tool – which we call MacroGreen – is easily produced by expression in Escherichia coli, and can detect both mono-and poly-ADP-ribosylation of diverse proteins in vitro. Staining with MacroGreen allows detection of ADP-ribosylation at sites of DNA damage by fluorescence microscopy. MacroGreen can also be used to quantify modification of target proteins in overlay assays, and to screen for PARP inhibitors in high-throughput format with excellent assay statistics. We expect that this broadly applicable tool will facilitate ADP-ribosylation related discoveries, including by laboratories that do not specialize in this field. Nature Publishing Group UK 2021-07-28 /pmc/articles/PMC8319303/ /pubmed/34321589 http://dx.doi.org/10.1038/s42003-021-02439-w Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Comment
García-Saura, Antonio Ginés
Herzog, Laura K.
Dantuma, Nico P.
Schüler, Herwig
MacroGreen, a simple tool for detection of ADP-ribosylated proteins
title MacroGreen, a simple tool for detection of ADP-ribosylated proteins
title_full MacroGreen, a simple tool for detection of ADP-ribosylated proteins
title_fullStr MacroGreen, a simple tool for detection of ADP-ribosylated proteins
title_full_unstemmed MacroGreen, a simple tool for detection of ADP-ribosylated proteins
title_short MacroGreen, a simple tool for detection of ADP-ribosylated proteins
title_sort macrogreen, a simple tool for detection of adp-ribosylated proteins
topic Comment
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8319303/
https://www.ncbi.nlm.nih.gov/pubmed/34321589
http://dx.doi.org/10.1038/s42003-021-02439-w
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