Detecting Poly (ADP-Ribose) In Vitro and in Cells Using PAR Trackers.

ADP-ribosylation (ADPRylation) is a reversible posttranslational modification resulting in the covalent attachment of ADP-ribose (ADPR) moieties on substrate proteins. Naturally occurring protein motifs and domains, including WWEs, PBZs (PAR binding zinc fingers), 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 the preparation and use of poly(ADP-ribose) (PAR) Trackers (PAR-Ts)-optimized dimerization-dependent or split-protein reassembly PAR sensors containing a naturally occurring PAR binding domain fused to both halves of dimerization-dependent GFP (ddGFP) or split nano luciferase (NanoLuc), respectively. We also describe how these tools can be used for the detection and quantification of PAR levels in biochemical assays with extracts and in living cells. These protocols will allow users to explore the broad utility of PAR-Ts for detecting PAR in various experimental and biological systems.

Challa S ，Whitaker AL ，Kraus WL 《-》

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. 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.

Challa S ，Ryu KW ，Whitaker AL ，Abshier JC ，Camacho CV ，Kraus WL ... -  《eLife》

Generating Protein-Linked and Protein-Free Mono-, Oligo-, and Poly(ADP-Ribose) In Vitro.

Lin KY ，Huang D ，Kraus WL 《-》

Development and characterization of recombinant ADP-ribose binding reagents that allow simultaneous detection of mono and poly ADP-ribose.

ADP-ribosylation (ADPRylation) is a post-translational modification (PTM) of proteins mediated by the activity of a variety of ADP-ribosyltransferase (ART) enzymes, such as the Poly (ADP-ribose) Polymerase (PARP) family of proteins. This PTM is diverse in both form and biological functions, which makes it a highly interesting modification, but difficult to study due to limitations in reagents available to detect the diversity of ADPRylation. Recently we developed a set of recombinant antibody-like ADP-ribose (ADPR) binding proteins using naturally occurring ADPR binding domains (ARBDs), including macrodomains and WWE domains, functionalized by fusion to the constant "Fc" region of rabbit immunoglobulin. Herein, we present an expansion of this biological toolkit, where we have replaced the rabbit Fc sequence with the sequence from two other species, mouse and goat. These new reagents are based on a previously characterized set of naturally occurring ARBDs with known specificity. Characterization of the new reagents demonstrates that they can be detected in a species-dependent manner by secondary immunological tools, recognize specific ADPR moieties, and can be used for simultaneous detection of mono ADPR and poly ADPR at single-cell resolution in various antibody-based assays. The expansion of this toolkit will allow for more multiplexed assessments of the complexity of ADPRylation biology in many biological systems.

Chiu SP ，Camacho CV ，Kraus WL 《-》

Biochemical and Biophysical Assays of PAR-WWE Domain Interactions and Production of iso-ADPr for PAR-Binding Analysis.

Wang Z ，Xu W 《-》