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

Sridevi Challa, Amy L Whitaker, W Lee Kraus

Journal: Methods in molecular biology (Clifton, N.J.) 2022;2609():75-90

PMID: 36515830

Abstract

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.

Address: Laboratory of Signaling and Gene Regulation, Cecil H. and Ida Green Center for Reproductive Biology Sciences, The University of Texas Southwestern Medical Center, Dallas, TX, USA. [email protected].; Laboratory of Signaling and Gene Regulation, Cecil H. and Ida Green Center for Reproductive Biology Sciences, The University of Texas Southwestern Medical Center, Dallas, TX, USA.; Program in Genetics, Development, and Disease, Graduate School of Biomedical Sciences, University of Texas Southwestern Medical Center, Dallas, TX, USA.; Laboratory of Signaling and Gene Regulation, Cecil H. and Ida Green Center for Reproductive Biology Sciences, The University of Texas Southwestern Medical Center, Dallas, TX, USA. [email protected].

DOI: 10.1007/978-1-0716-2891-1_5

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MeSH Terms: Poly Adenosine Diphosphate Ribose, Ribose, Adenosine Diphosphate Ribose, ADP-Ribosylation, Protein Processing, Post-Translational, Recombinant Proteins