Development and characterization of new tools for detecting poly(ADP-ribose) in vitro and in vivo
Sridevi Challa,
Keun W Ryu,
Amy L Whitaker,
Jonathan C Abshier,
Cristel V Camacho,
W Lee Kraus
Affiliations
Sridevi Challa
Laboratory of Signaling and Gene Regulation, Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, United States; Division of Basic Research, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, United States
Keun W Ryu
Laboratory of Signaling and Gene Regulation, Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, United States; Division of Basic Research, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, United States
Amy L Whitaker
Laboratory of Signaling and Gene Regulation, Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, United States; Division of Basic Research, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, United States; Program in Genetics, Development, and Disease, Graduate School of Biomedical Sciences, University of Texas Southwestern Medical Center, Dallas, United States
Jonathan C Abshier
Laboratory of Signaling and Gene Regulation, Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, United States; Division of Basic Research, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, United States
Laboratory of Signaling and Gene Regulation, Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, United States; Division of Basic Research, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, United States
Laboratory of Signaling and Gene Regulation, Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, United States; Division of Basic Research, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, United States
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.
