Identification of protein substrates of specific PARP enzymes using analog-sensitive PARP mutants and a “Clickable” NAD+ analog

Bryan A. Gibson, W. Lee Kraus

Research output: Chapter in Book/Report/Conference proceedingChapter

5 Scopus citations

Abstract

The PARP family of ADP-ribosyl transferases contains 17 members in human cells, most of which catalyze the transfer of the ADP-ribose moiety of NAD+ onto their target proteins. This posttranslational modification plays important roles in cellular signaling, especially during cellular stresses, such as heat shock, inflammation, unfolded protein responses, and DNA damage. Knowing the specific proteins that are substrates for individual PARPs, as well as the specific amino acid residues in a given target protein that are ADP-ribosylated, is a key step in understanding the biology of individual PARPs. Recently, we developed a robust NAD+ analog-sensitive approach for PARPs, which allows PARP-specific ADP-ribosylation of substrates that is suitable for subsequent copper-catalyzed azide-alkyne cycloaddition (“click chemistry”) reactions. When coupled with proteomics and mass spectrometry, the analog-sensitive PARP approach can be used to identify the specific amino acids that are ADP-ribosylated by individual PARP proteins. In this chapter, we describe the key facets of the experimental design and application of the analog-sensitive PARP methodology to identify site-specific modification of PARP target proteins.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages111-135
Number of pages25
Volume1608
DOIs
StatePublished - 2017

Publication series

NameMethods in Molecular Biology
Volume1608
ISSN (Print)1064-3745

Keywords

  • ADP-ribosylation
  • Analog-sensitivity
  • Automodification
  • Click chemistry
  • Mono(ADP-ribosyl)ation
  • Mutation
  • NAD analog
  • PARP
  • Poly(ADP-ribose) polymerase
  • Poly(ADP-ribosyl)ation
  • Posttranslational modification

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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    Gibson, B. A., & Kraus, W. L. (2017). Identification of protein substrates of specific PARP enzymes using analog-sensitive PARP mutants and a “Clickable” NAD+ analog. In Methods in Molecular Biology (Vol. 1608, pp. 111-135). (Methods in Molecular Biology; Vol. 1608). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-6993-7_9