Elucidation of the roles of the poly(ADP-ribose)polymerases PARP-1 and PARP-2 by RNA interference

Abstract

Poly(ADP-ribose)polymerase-1 (PARP-1) and PARP-2 belong to a family of related proteins that play diverse roles in various cellular functions, and share a highly homologous catalytic domain with a common enzymatic activity: the synthesis of protein-bound ADP-ribose polymers of various sizes (up to 200 units) and complexities (linear or branched). This modification is a dynamic process, as indicated by the short half-life of the ADP-ribose polymer, which is subject to degradation by poly(ADP-ribose)gylcohydrolase (PARG) [1]. PARP-1 has been initially thought to be the only existing enzyme with poly(ADP-ribosyl)ating activity in mammalian cells. However, this view has been challenged by the identification of novel poly(ADP-ribosyl)ating enzymes [2-4]. PARP-1 plays a multifunctional role in many cellular processes, including DNA repair, recombination, cell proliferation, cell death and contributes to the maintenance of genomic stability. On the other hand, excessive PARP-1 activation has been involved in the pathogenesis of various cardiovascular and inflammatory diseases. Compared with other members of the PARP family, PARP-2 bears the strongest resemblance to PARP-1. So far, PARP-1 and PARP-2 are the sole members, whose catalytic activity is stimulated in vitro and in vivo by DNA strand breaks, suggesting that they are both involved in the cellular response to DNA damage [3,5,6]. Further functional similarities of PARP-2 with PARP-1 involve: i) interaction with components of the BER machinery [6]; ii) involvement in the repair of DNA alkylation or γ-irradiation damage [7]; and iii) participation in telomere length control [8,9]. On the other hand, cumulating evidence points to non-redundant functions of the two enzymes [10]. For instance, although both PARP-1 and PARP-2 localize to active centromeres, the former exhibit a broader distribution extending to the pericentromeric region, while the latter appears to be more specific to centromeric chromatin where it localizes as discrete paired dots [11]. Loss of parp-2 gene function has also been linked to a specific instability of the X chromosome in females, manifested as female embryonic lethality of parp-1+/-parp-2-/- mice and confirmed by cytologic analyses [7]. In addition, PARP-2 has been shown to form more stable complexes with telomeric protein TRF-2 than PARP-1. Indeed, PARP-2 is able to ADP-ribosylate TRF-2 in vitro thus regulating its binding to DNA [8]. Finally, specific functions of PARP-2, but not PARP-1, have been identified in various differentiation processes, including adipogenesis, spermatogenesis and T lymphocyte development [12-14]. However, owing to the presence of additional PARPs and to the lethal phenotype of parp-1-/-parp-2-/- double mutants, the specific roles of these two PARP enzymes had not been clarified. The main goal of the present thesis was to study the roles of PARP-1 and PARP-2 in alkylation-induced cell death by means of RNA interference mediated gene silencing of the two proteins. We wanted to find out which of the two PARPs contributes to the array of poly(ADP-ribose) molecules (PARs) that act as a cell death signal by triggering the translocation of apoptosis-inducing factor (AIF), a mitochondrial protein that acts downstream of PAR. Furthermore, the effects of PARP-1 and PARP-2 on major upstream signals of the apoptosis pathway were investigated in the absence and presence of DNA damage by TaqMan Low Density Array analyses.