| Chapter title |
Identification of Protein Substrates of Specific PARP Enzymes Using Analog-Sensitive PARP Mutants and a “Clickable” NAD+ Analog
|
|---|---|
| Chapter number | 9 |
| Book title |
Poly(ADP-Ribose) Polymerase
|
| Published in |
Methods in molecular biology, July 2017
|
| DOI | 10.1007/978-1-4939-6993-7_9 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-6992-0, 978-1-4939-6993-7
|
| Authors |
Bryan A. Gibson, W. Lee Kraus, Gibson, Bryan A., Kraus, W. Lee |
| 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. |
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Demographic breakdown
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Mendeley readers
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|---|---|---|
| Unknown | 21 | 100% |
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|---|---|---|
| Researcher | 4 | 19% |
| Student > Ph. D. Student | 3 | 14% |
| Student > Bachelor | 2 | 10% |
| Professor > Associate Professor | 2 | 10% |
| Unspecified | 2 | 10% |
| Other | 4 | 19% |
| Unknown | 4 | 19% |
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| Unspecified | 2 | 10% |
| Agricultural and Biological Sciences | 2 | 10% |
| Immunology and Microbiology | 1 | 5% |
| Other | 0 | 0% |
| Unknown | 5 | 24% |