Investigation of PARP-1, PARP-2, and PARG interactomes by affinity-purification mass spectrometry

Abstract

Background: Poly(ADP-ribose) polymerases (PARPs) catalyze the formation of poly(ADP-ribose) (pADPr), a post-translational modification involved in several important biological processes, namely surveillance of genome integrity, cell cycle progression, initiation of the DNA damage response, apoptosis, and regulation of transcription. Poly(ADP-ribose) glycohydrolase (PARG), on the other hand, catabolizes pADPr and thereby accounts for the transient nature of poly(ADP-ribosyl)ation. Our investigation of the interactomes of PARP-1, PARP-2, and PARG by affinity-purification mass spectrometry (AP-MS) aimed, on the one hand, to confirm current knowledge on these interactomes and, on the other hand, to discover new protein partners which could offer insights into PARPs and PARG functions.

Results: PARP-1, PARP-2, and PARG were immunoprecipitated from human cells, and pulled-down proteins were separated by gel electrophoresis prior to in-gel trypsin digestion. Peptides were identified by tandem mass spectrometry. Our AP-MS experiments resulted in the identifications of 179 interactions, 139 of which are novel interactions. Gene Ontology analysis of the identified protein interactors points to five biological processes in which PARP-1, PARP-2 and PARG may be involved: RNA metabolism for PARP-1, PARP-2 and PARG; DNA repair and apoptosis for PARP-1 and PARP-2; and glycolysis and cell cycle for PARP-1.

Conclusions: This study reveals several novel protein partners for PARP-1, PARP-2 and PARG. It provides a global view of the interactomes of these proteins as well as a roadmap to establish the systems biology of poly(ADP-ribose) metabolism.

Figure 1

Venn diagram illustrating the number of interactors identified by AP-MS for PARP-1, PARP-2 and PARG. Numbers outside the overlaps correspond to proteins unique to either the PARP-1, PARP-2 or PARG immunoprecipitation dataset whereas numbers inside the overlaps correspond to proteins common to two or three datasets.

Figure 2

Complementary western blot analysis of novel PARP-1 and PARP-2 interactors. A and B) Immunoprecipitation of PARP-1 and associated proteins. In A) immunodetection of PARP-1 (bait) and the novel interactors Btf, identified by AP-MS. In B) immunodetection of novel PARP-1 interactors FMR-1 and AIF, absent from AP-MS. C and D) Immunoprecipitation of PARP-2 and associated proteins. In A) immunodetection of PARP-2 (bait) and the novel interactors KU70, KU80 and FMR-1, identified by AP-MS. In B) immunodetection of novel PARP-2 interactors BTF, AIF and STAT-1, absent from AP-MS. Each immunodetection is done in whole cellular extract (WCE) and immunoprecipitates (IP) from control (CTRL) and the bait (PARP-1 or FLAG-PARP-2).

Figure 3

Gene Ontology analysis of PARP-2 interactors. Gene Ontology classification by biological process of the interactors of PARP-2 identified by AP-MS. See text for details.

Figure 4

Gene Ontology analysis of PARP-2 interactors. Gene Ontology classification by biological process of the interactors of PARP-2 identified by AP-MS. See text for details.

Figure 5

Gene Ontology analysis of PARG interactors. Gene Ontology classification by biological process of the interactors of PARG identified by AP-MS. See text for details.