Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Challa, Sridevi, Ryu, Keun W, Whitaker, Amy L, Abshier, Jonathan C, Camacho, Cristel V, Kraus, W Lee
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2022
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
_version_ 1784695398297763840
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
work_keys_str_mv AT challasridevi developmentandcharacterizationofnewtoolsfordetectingpolyadpriboseinvitroandinvivo
AT ryukeunw developmentandcharacterizationofnewtoolsfordetectingpolyadpriboseinvitroandinvivo
AT whitakeramyl developmentandcharacterizationofnewtoolsfordetectingpolyadpriboseinvitroandinvivo
AT abshierjonathanc developmentandcharacterizationofnewtoolsfordetectingpolyadpriboseinvitroandinvivo
AT camachocristelv developmentandcharacterizationofnewtoolsfordetectingpolyadpriboseinvitroandinvivo
AT krauswlee developmentandcharacterizationofnewtoolsfordetectingpolyadpriboseinvitroandinvivo