TY - CHAP
T1 - Assay for Protein Modification by Poly-ADP-Ribose In Vitro
AU - Olabisi, Opeyemi A.
AU - Chow, Chi Wing
N1 - Publisher Copyright:
© 2011, Springer Science+Business Media, LLC.
PY - 2011
Y1 - 2011
N2 - The enzymatic function of poly(adenosine diphosphate (ADP)-ribose) polymerase (PARP) is central to many of its function as a component of DNA repair machinery, modulator of gene transcription, and cell differentiation. While the auto-modification domain of PARP has been shown to be a primary acceptor site of poly-ADP ribose (pADPr), other DNA binding nuclear proteins are also modified by pADPr. It is generally accepted that pADPr polymer is built upon the carboxyl side chain of specific Glu, Asp, and/or Lys residues within the target protein. Identification of the unique amino acid acceptors of pADPr in these nuclear proteins is an active area of study. Because of the heterogeneity of pADPr chain on modified protein targets, the resulting modified proteins have unpredictable final masses, making it difficult to identify acceptor amino acids. Using recombinant proteins, in vitro pADP ribosylation assay and mass spectrometry, we have been able to identify conserved Glu residue in transcription factor NFAT that is enzymatically modified in vitro with pADPr by PARP-1. We discuss this protocol here as a model approach for identifying pADPr acceptors in other nuclear proteins.
AB - The enzymatic function of poly(adenosine diphosphate (ADP)-ribose) polymerase (PARP) is central to many of its function as a component of DNA repair machinery, modulator of gene transcription, and cell differentiation. While the auto-modification domain of PARP has been shown to be a primary acceptor site of poly-ADP ribose (pADPr), other DNA binding nuclear proteins are also modified by pADPr. It is generally accepted that pADPr polymer is built upon the carboxyl side chain of specific Glu, Asp, and/or Lys residues within the target protein. Identification of the unique amino acid acceptors of pADPr in these nuclear proteins is an active area of study. Because of the heterogeneity of pADPr chain on modified protein targets, the resulting modified proteins have unpredictable final masses, making it difficult to identify acceptor amino acids. Using recombinant proteins, in vitro pADP ribosylation assay and mass spectrometry, we have been able to identify conserved Glu residue in transcription factor NFAT that is enzymatically modified in vitro with pADPr by PARP-1. We discuss this protocol here as a model approach for identifying pADPr acceptors in other nuclear proteins.
KW - Poly(adenosine diphosphate (ADP)-ribose) polymerase (PARP)
KW - Poly-ADP ribose (pADPr)
KW - Transcription factor NFAT
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U2 - 10.1007/978-1-61779-270-0_3
DO - 10.1007/978-1-61779-270-0_3
M3 - Chapter
C2 - 21870253
AN - SCOPUS:80054723501
SN - 9781617792694
T3 - Methods in Molecular Biology
SP - 47
EP - 55
BT - Poly(ADP-ribose) Polymerase
PB - Humana Press Inc.
ER -