Abstract

Recent findings have highlighted the fact that the regulation of poly(ADP-ribose) (pADPr) metabolism is not only important for DNA repair but is also involved in apoptosis signaling and for the regulation of circadian rhythms [1, 2]. To gain insight into the cellular pathways in which PARPs and pADPr are involved, we have initiated a study to identify pADPr-binding proteins as well as protein interactors of three nuclear PARPs, PARP-1, PARP-2 and PARP-3, using a proteomic approach. To identify pADPr-binding proteins, Rotofor fractions of a HeLa cellular extract were screened with 32P-labeled pADPr using a protein blot assay. The most abundant pADPr-binding proteins were identified by peptide mass finger printing as hnRNPs, a family of proteins that bind pre-mRNA into functional complexes involved in mRNA maturation and transport to the cytoplasm. PARP interactors were isolated by immunoprecipitations. Endogenous PARP-1 was immunoprecipitated from HeLa S3 nuclei using the PARP-1 specific antibodies C2-10, F1-23 and 96-72. PARP-2 and PARP-3, transiently expressed in COS-7 cells as N-terminal fusions with the FLAG epitope, were immunoprecipitated with the M2 anti-FLAG antibody. Immunoprecipitated proteins digested with trypsin were identified by mass spectrometry and Western analysis. This approach identified previously reported PARP-1 interactors such as topoisomerase I, histones H1, H3 and H4, hnRNPs and nucleolin, thus confirming the interactions between these proteins and PARP-1 and validating our methodology. New interactions were also found, in particular with nucleolar proteins such as EBNA1-binding protein. The analysis of PARP-2 and PARP-3 interactors is ongoing and results of this analysis will be presented.References: 1.Yu SW, Wang H, Poitras MF, Coombs C et al: Mediation of Poly(ADP-Ribose) Polymerase-1 Dependent Cell Death by Apoptosis Inducing Factor. Science, 2002; 297: 259-263 2.Panda S, Poirier GG, Kay SA: tej defines a role for poly(ADP-ribosyl)ation in establishing period length of the Arabidopsis circadian oscillator. Developmental Cell, 2002; 3(1): 51-61 3.Gagné JP, Hunter J, Labreque B et al: A proteomic approach to the identification of heterogeneous nuclear ribonucleoproteins as a new family of poly(ADP-ribose)-binding proteins. Biochemical J, 2003; In press

Keywords: proteomic, PARP interactors, poly(ADP-ribose) binding proteins, Mass Spectrometry