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Related Experiment Video

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

Sridevi Challa1, Amy L Whitaker2,3, W Lee Kraus4

  • 1Laboratory 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. sridevi.challa@utsouthwestern.edu.

Methods in Molecular Biology (Clifton, N.J.)
|December 14, 2022
PubMed
Summary

Researchers developed novel poly(ADP-ribose) (PAR) Trackers (PAR-Ts) for detecting ADP-ribosylation (ADPRylation). These sensors enable dynamic monitoring of PAR levels in biochemical assays and living cells, advancing ADPRylation research.

Keywords:
ADP-ribose (ADPR)ADP-ribosyl transferase (ART)ADP-ribosylation (ADPRylation)ADPR binding domain (ARBD)AutomodificationDimerization-dependent green fluorescent protein (ddGFP)Live cell imagingNano luciferase (NanoLuc)Poly(ADP-ribosyl)ation (PARylation)Posttranslational modification (PTM)

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • ADP-ribosylation (ADPRylation) is a crucial reversible posttranslational modification involving ADP-ribose (ADPR) attachment to proteins.
  • Protein motifs like WWEs, PBZs, and macrodomains function as ADPR "readers."
  • Existing ADPR detection reagents, while useful, struggle to capture the dynamic nature of ADPRylation.

Purpose of the Study:

  • To develop novel, dynamic sensors for detecting and quantifying poly(ADP-ribose) (PAR).
  • To create tools that overcome limitations of current antibody-like ADPR detection reagents.
  • To enable the study of ADPRylation dynamics in various biological contexts.

Main Methods:

  • Preparation of poly(ADP-ribose) (PAR) Trackers (PAR-Ts).
  • PAR-Ts utilize optimized dimerization-dependent or split-protein reassembly systems (ddGFP, NanoLuc).
  • PAR-Ts incorporate naturally occurring PAR binding domains fused to sensor components.

Main Results:

  • Successful preparation and validation of PAR-Ts for ADPR detection.
  • Demonstrated utility of PAR-Ts in biochemical assays using cell extracts.
  • Showcased the application of PAR-Ts for monitoring PAR levels in living cells.

Conclusions:

  • PAR-Ts provide a novel and effective method for detecting and quantifying PAR.
  • These sensors offer enhanced capabilities for studying the dynamic nature of ADPRylation.
  • PAR-Ts are versatile tools applicable to diverse experimental and biological systems.