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Development and characterization of new tools for detecting poly(ADP-ribose) in vitro and in vivo
ADP-ribosylation (ADPRylation) is a reversible post-translation modification resulting in the covalent attachment of ADP-ribose (ADPR) moieties on substrate proteins. Naturally occurring protein motifs and domains, including WWEs, PBZs, and macrodomains, act as ‘readers’ for protein-linked ADPR. Alt...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
eLife Sciences Publications, Ltd
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9045816/ https://www.ncbi.nlm.nih.gov/pubmed/35476036 http://dx.doi.org/10.7554/eLife.72464 |
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author | Challa, Sridevi Ryu, Keun W Whitaker, Amy L Abshier, Jonathan C Camacho, Cristel V Kraus, W Lee |
author_facet | Challa, Sridevi Ryu, Keun W Whitaker, Amy L Abshier, Jonathan C Camacho, Cristel V Kraus, W Lee |
author_sort | Challa, Sridevi |
collection | PubMed |
description | ADP-ribosylation (ADPRylation) is a reversible post-translation modification resulting in the covalent attachment of ADP-ribose (ADPR) moieties on substrate proteins. Naturally occurring protein motifs and domains, including WWEs, PBZs, and macrodomains, act as ‘readers’ for protein-linked ADPR. Although recombinant, antibody-like ADPR detection reagents containing these readers have facilitated the detection of ADPR, they are limited in their ability to capture the dynamic nature of ADPRylation. Herein, we describe and characterize a set of poly(ADP-ribose) (PAR) Trackers (PAR-Ts)—optimized dimerization-dependent or split-protein reassembly PAR sensors in which a naturally occurring PAR binding domain, WWE, was fused to both halves of dimerization-dependent GFP (ddGFP) or split Nano Luciferase (NanoLuc), respectively. We demonstrate that these new tools allow the detection and quantification of PAR levels in extracts, living cells, and living tissues with greater sensitivity, as well as temporal and spatial precision. Importantly, these sensors detect changes in cellular ADPR levels in response to physiological cues (e.g., hormone-dependent induction of adipogenesis without DNA damage), as well as xenograft tumor tissues in living mice. Our results indicate that PAR Trackers have broad utility for detecting ADPR in many different experimental and biological systems. |
format | Online Article Text |
id | pubmed-9045816 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | eLife Sciences Publications, Ltd |
record_format | MEDLINE/PubMed |
spelling | pubmed-90458162022-04-28 Development and characterization of new tools for detecting poly(ADP-ribose) in vitro and in vivo Challa, Sridevi Ryu, Keun W Whitaker, Amy L Abshier, Jonathan C Camacho, Cristel V Kraus, W Lee eLife Biochemistry and Chemical Biology ADP-ribosylation (ADPRylation) is a reversible post-translation modification resulting in the covalent attachment of ADP-ribose (ADPR) moieties on substrate proteins. Naturally occurring protein motifs and domains, including WWEs, PBZs, and macrodomains, act as ‘readers’ for protein-linked ADPR. Although recombinant, antibody-like ADPR detection reagents containing these readers have facilitated the detection of ADPR, they are limited in their ability to capture the dynamic nature of ADPRylation. Herein, we describe and characterize a set of poly(ADP-ribose) (PAR) Trackers (PAR-Ts)—optimized dimerization-dependent or split-protein reassembly PAR sensors in which a naturally occurring PAR binding domain, WWE, was fused to both halves of dimerization-dependent GFP (ddGFP) or split Nano Luciferase (NanoLuc), respectively. We demonstrate that these new tools allow the detection and quantification of PAR levels in extracts, living cells, and living tissues with greater sensitivity, as well as temporal and spatial precision. Importantly, these sensors detect changes in cellular ADPR levels in response to physiological cues (e.g., hormone-dependent induction of adipogenesis without DNA damage), as well as xenograft tumor tissues in living mice. Our results indicate that PAR Trackers have broad utility for detecting ADPR in many different experimental and biological systems. eLife Sciences Publications, Ltd 2022-04-27 /pmc/articles/PMC9045816/ /pubmed/35476036 http://dx.doi.org/10.7554/eLife.72464 Text en © 2022, Challa et al https://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited. |
spellingShingle | Biochemistry and Chemical Biology Challa, Sridevi Ryu, Keun W Whitaker, Amy L Abshier, Jonathan C Camacho, Cristel V Kraus, W Lee Development and characterization of new tools for detecting poly(ADP-ribose) in vitro and in vivo |
title | Development and characterization of new tools for detecting poly(ADP-ribose) in vitro and in vivo |
title_full | Development and characterization of new tools for detecting poly(ADP-ribose) in vitro and in vivo |
title_fullStr | Development and characterization of new tools for detecting poly(ADP-ribose) in vitro and in vivo |
title_full_unstemmed | Development and characterization of new tools for detecting poly(ADP-ribose) in vitro and in vivo |
title_short | Development and characterization of new tools for detecting poly(ADP-ribose) in vitro and in vivo |
title_sort | development and characterization of new tools for detecting poly(adp-ribose) in vitro and in vivo |
topic | Biochemistry and Chemical Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9045816/ https://www.ncbi.nlm.nih.gov/pubmed/35476036 http://dx.doi.org/10.7554/eLife.72464 |
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