COINVOLGIMENTO DEL DOMINIO BRCT DI PARP1 NELLA VIA APOPTOTICA p53 DIPENDENTE INDOTTA DA RAGGI UV

Poly(ADP-ribose) polymerase-1 (PARP-1) is one of the most abundant proteins within mammalian cells. PARP-1 is an abundant nuclear enzyme involved in DNA repair and transcriptional regulation and is now recognized as a key regulator of cell survival and cell death as well as a master component of a number of transcription factors involved in tumor development and inflammation. PARP-1 is essential to the repair of DNA single-strand breaks via the base excision repair pathway. Despite many interesting studies that implicate PARP-1 in transcription, chromatin remodelling, apoptosis, DNA repair and several neurological disorders, its precise role is still unclear. The aim of my studies is represented by the involvement of the PARP-1 domain, BRCT in the induction of p53-dependent apoptotic pathway. The p53 protein is one of the most important tumor suppressor proteins. Normally, the p53 protein is in a latent state. However, in case of DNA damage, nucleotide depletion or hypoxia ,when its activity is required, p53 becomes rapidly activated and initiates transcription of pro-apoptotic and cell cycle arrest-inducing target genes. By means of the aid of T7 phage display have been the expression of a library of peptides able to bind Phosphopeptides; through biopanning and after several cycle of selection 18 clones were isolated of which 9 phosphopeptides bound p53, but not able to bind non phosphorilated p53. The clones obtained were then amplified and further selected with an electrophoretic run; sequenced and the subsequent alignment of the sequences with those of protein database SwissProt: 2 clones (S4p5311 and S4p5316) results correspond to PARP-1 protein sequences, BRCT domain. Therefore we developed a model of cell death, characterized by cells U2OS (Human Bone Osteosarcoma) treated with UV and after 24 hours of exposure we obtained a mortality of 50-60%. We then analyzed the morphological and immunochemistry markers that confirmed, our hypothesis, Death by UV irradiation happen by apoptosis; final confirmation is given to us by the study of cell vitality by colorimetric assay. Moreover we have studied how changes in the expression of p53 protein following treatment with UV, revealing an increase in time-dependent beginning 3 hours after treatment and with a maximum to 24 hours. Furthermore by Affinity Purification, performed with functionalized beads with GST-BRCT group were incubated with cell lysates from cultures at different times of treatment. BRCT-p53 interaction occurs with a very different kinetics from the expression of p53. We supposed that the key event which regulates the interaction between p53 and BRCT is a phosphorylation and this interaction is able to reduce cell death after exposure to UV. To test whether the BRCT-p53 interaction could also occur within cells, we transfected some culture cell by using Lipofectamine, with two separate vectors. One, able to express a GFP and the other BRCT-GFP fusion protein. Then using immunoprecipitation technique we were able to verify the interaction occurred within the cell, between BRCT and p53, and we discovered a protective effect against apoptosis induced by UV radiation. We didn’t see the same effect on the cells transfected only with the GFP vector, and again on a different line cells called H1299 which is p3 negative In conclusion, we have validated the model of cell death developed by us for apoptosis experiment and we have characterized the interaction between BRCT and p53 in several biological condition. For the future we would like to discover if the phosphorylation represent the main even that regulates the interaction between p53 and BRCT, and how this mechanism performs its protective effect.