Identification of candidate substrates for poly(ADP-ribose) polymerase-2 (PARP2) in the absence of DNA damage using high-density protein microarrays

Abstract

Poly(ADP-ribose) polymerase-2 (PARP2) belongs to the ADP-ribosyltransferase family of enzymes that catalyze the addition of ADP-ribose units to acceptor proteins, thus affecting many diverse cellular processes. In particular, PARP2 shares with PARP1 and, as recently highlighted, PARP3 the sole property of being catalytically activated by DNA-strand breaks, implying key downstream functions in the cellular response to DNA damage for both enzymes. However, evidence from several studies suggests unique functions for PARP2 in additional processes, possibly mediated through its basal, DNA-damage unstimulated ADP-ribosylating activity. Here, we describe the development and application of a protein microarray-based approach tailored to identify proteins that are ADP-ribosylated by PARP2 in the absence of DNA damage mimetics and might thus represent useful entry points to the exploration of novel PARP2 functions. Several candidate substrates for PARP2 were identified and global hit enrichment analysis showed a clear enrichment in translation initiation and RNA helicase molecular functions. In addition, the top scoring candidates FK506-binding protein 3 and SH3 and cysteine-rich domain-containing protein 1 were selected and confirmed in a complementary assay format as substrates for unstimulated PARP2. Structured digital abstract A list of the large number of protein-protein interactions described in this article is available via the MINT article ID PARP2 ADP-ribosyltransferase activity is stimulated by DNA damage to participate in the DNA repair process, but there is also evidence for unique functions mediated through basal, DNA-damage independent enzyme activity. We describe a protein microarray approach tailored to identify PARP2 substrates in the absence of DNA damage mimetics as potential exploratory entry points and report FKBP3 and STAC1 as top-scoring candidates. © 2011 FEBS.