Identification of the ADP-Ribosylation Sites in the PARP-1 Automodification Domain: Analysis and Implications

Abstract

Poly(ADP-ribose) polymerase-1 (PARP-1) is a multimodular (domains A, B, C, D, E, and F) nuclear protein that participates in many fundamental cellular activities. Stimulated by binding to nicked DNA, PARP-1 catalyzes poly(ADP-ribosyl)ation of the acceptor proteins and itself using NAD(+) as a substrate. Early studies suggested that domain D is likely an interface for protein-protein interaction between PARP-1 and its targets and is also the primary region for automodification. However, determination of the modification sites has been complicated by the heterogeneous nature of the poly(ADP-ribose) polymer. Here we report a strategy to identify the modification sites on domain D using the PARP-1 E988Q mutant, which only catalyzes mono(ADP-ribosyl)ation. Trypsin digestion of the modified domain D followed by LC-MS/MS analysis led to the identification of three ADP-ribosylation sites in domain D (D387, E488, and E491). Our data also show, in contrast to early reports, that automodification of PARP-1 is not limited to domain D but occurs beyond this region. In addition, domain D is not essential for PARP-1 activity since PARP-1 mutant having domain D deleted is still catalytically active. Two synthetic peptides with amino acid sequences derived from the ADP-ribosylation sites of domain D were also demonstrated to act as PARP-1 substrates. The methodology and the results reported herein will facilitate future studies of PARP-1 catalysis.

PARP-3 Is a Mono-ADP-ribosylase That Activates PARP-1 in the Absence of DNA

“…Mutational analysis in the work of Altmeyer et al (23) led to the identification of three lysine residues in the PARP-1 auto-modification domain (Lys 498 , Lys 521 , and Lys 524 ) as the sites of ADP-ribosylation. Recently, the first successful identification of the ADPribosylation sites in PARP-1 was done by mass spectrometry (27). In this study, the glutamic and aspartic acid residues in the PARP-1 auto-modification domain (Glu 488 , Glu 491 , and Asp 387 ) were identified as the acceptor amino acid residues.…”

Signaling Mechanism of Poly(ADP-Ribose) Polymerase-1 (PARP-1) in Inflammatory Diseases

“…[22][23][24] Dimerization is assumed to be a prerequisite for PARP-1 activation 25 ; however, the AMD deletion mutant is catalytically active, indicating that this segment is not indispensable for PARP-1 activity. 20 Other studies 24,25 have suggested that interaction in the N-terminal region is required for PARP-1 dimerization and activation. The structural basis and significance of PARP-1's dimerization for its catalytic activity are not yet clear.…”

“…There are several glutamic acid residues in this domain that are suggested to be the site for covalent binding with poly(ADP-ribose) on PARP-1 activation. 19,20 However, other groups argue that individual lysine residues, not glutamic acid, serve as acceptor sites for ADP ribosylation in the AMD. 21 The PAR modification of glutamic residues in the NAD ϩ -binding domain and the DBD has also been noted.…”

“…21 The PAR modification of glutamic residues in the NAD ϩ -binding domain and the DBD has also been noted. 19,20 Irrespective of the site, automodification is accepted as a mechanism for regulating PARP-1 activity and control PAR synthesis 19 ; however, the exact impact and mechanisms of automodification on enzyme function need further investigation.…”

“…26,27 The influence of the automodification of the glutamic acid residues within this domain on PARP-1's catalytic activity has not yet been fully addressed. 19,20 In addition to DNA breaks, DNA hairpins, cruciform, and stably unpaired regions have all been considered effective determinants of PARP-1 activation. 28,29 After its binding to DNA, PARP-1 catalyzes the formation of PARs.…”