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Title: Nibrin and promyelocytic leukemia proteins in sensing and signaling of the dna double strand breaks
Authors: Cilli, Domenica
metadata.dc.contributor.advisor: Di Masi, Alessandra
Issue Date: 16-Feb-2015
Publisher: Università degli studi Roma Tre
Abstract: The DNA double-strand breaks (DSBs) are among the most cytotoxic types of DNA damage, which if left unrepaired can lead to mutations or gross chromosomal aberrations and promote the onset of diseases associated with genomic instability, such as cancer. One of the most discernible hallmarks of the cellular response to DSBs is the accumulation and local concentration of a plethora of DNA damage signalling and repair proteins in the vicinity of the lesion, initiated by the ATM-mediated phosphorylation of H2AX at Ser139 (the phosphorylated H2AX protein being named γ-H2AX), the recruitment of the macromolecular complex made up of MRE11, RAD50 and nibrin (also named NBN) (MRN), and the generation of distinct nuclear compartments called ionizing radiation (IR)-induced foci (IRIF). The assembly of proteins at the DSB-flanking chromatin occurs in a highly ordered and strictly hierarchical fashion. To a large extent, this is achieved by regulation of protein-protein interactions triggered by a variety of post-translational modifications, including phosphorylation. Nibrin is composed of three regions: the N-terminal region contains the FHA domain, and two BRCT tandem domains (tBRCT); the central region, contains Ser residues which are phosphorylated by ATM kinase in response to IR; and the C-terminal region, containing the MRE11-binding domain and the ATM-binding motif. The tBRCT domains, present in more than 50 proteins involved in the DDR and in cell cycle control, are important mediators of phosphorylation-dependent protein-protein interactions in processes related to cell cycle checkpoint and DNA repair functions. Mutations at the homozygous or compound heterozygous status within the nibrin coding gene (NBN) are responsible for the Nijmegen breakage syndrome (NBS; OMIM #251260), a rare genetic disorder characterized by an autosomic recessive inheritance, whose signs are, among others, microcephaly, immunodeficiency, spontaneous chromosomal instability, and sensitivity to IR. The majority of NBS patients are homozygous for the 657del5 hypomorphic mutation. This mutation splits up the tBRCT domain and determines the synthesis of two fragments with molecular weights of 26 and 70 kDa. The 26 kDa fragment includes the FHA and the BRCT1 domains, while the 70 kDa fragment contains the BRCT2 domain and the C-terminal region. A further pathogenic mutation localized within the nibrin tBRCT domains is the Arg215Trp substitution. Remarkably, both the 657del5 and Arg215Trp NBN mutations are responsible for cancer susceptibility both at the homozygous and heterozygous status. The aim of the first year of this PhD project was to shed light of the role of nibrin tBRCT domains in the DSB sensing and signaling. Results obtained indicated that both mutations, although not altering the assembly of the MRN complex, affect the MRE11 IRIF formation and the DSB signaling impairing the phosphorylation of both ATM and ATM downstream targets (e.g., SMC1 and p53). We further demonstrated that the cleavage of the tBRCT domains of nibrin by the 657del5 mutation affects the DNA damage response less than the Arg215Trp mutation. Indeed, the 70 kDa NBN fragment, arising from the 657del5 mutation, maintains the capability to interact with MRE11 and γ-H2AX, as well as to form IRIF. Overall, data obtained indicated that the tBRCT domains of nibrin are involved in the proper activation of the DDR, altering the localization of repair proteins at the DSB, as well as the DNA damage signaling. Furthermore, we observed that the Arg215Trp mutation impairs γ-H2AX binding, suggesting the presence of a direct interaction between nibrin tBRCT and γ-H2AX. However, data obtained by literature are in contrast with this result, suggesting that nibrin- γ-H2AX interaction is indirect, being mediated by MDC1 protein.To further clarify this aspect, nibrin proteins carrying either naturaloccurring or artificial mutations (i.e., Arg215Trp and Lys160Met), all perturbing the relative geometry of the tBRCT domains, have been expressed, and spectrofluorimetric analyses have been performed. Results obtained seems to suggest that the mutations altering the putative tBRCT binding pocket also affect γ-H2AX binding, supporting the notion that the integrity of the tBRCT domains is pivotal for the proper interaction with the phosphorylated histone. Since nibrin has several important roles in the DDR, some of which resulting compromised by the pathogenic 657del5 homozygous mutation of NBN, during the second year of the PhD project an unsupervised proteomic approach, based upon strep-tag affinity chromatography, SDS-PAGE separation, and shotgun digestion of protein bands followed by MS/MS protein identification was performed. This allowed us to characterize the interactome of the full-length nibrin compared to that of the p26 and p70 fragments arising from the 657del5 mutation, both in presence and in absence of IR- induced DSBs. Results obtained indicate the occurrence of previously unreported protein interacting partners of the full-length nibrin and of the p26 fragment containing the FHA/BRCT1 domains, especially after cell irradiation. In particular, data shed light on new possible roles of nibrin in several biological processes as DSBs repair, cell cycle checkpoint activation, maintenance of chromosomal integrity, protein biosynthesis and antioxidant responses. Furthermore, we identified the interactors of the p26 fragment, which results involved in reactive oxygen species (ROS) scavenging, in the DDR, and in protein folding and degradation. In particular, we demonstrate that p26 interacts with PARP1 after IR, and this interaction exerts an inhibitory effect on PARP1 activity as measured by NAD+ levels. Furthermore, the p26-PARP1 interaction seems to be responsible for the persistence of ROS, and in turn of DSBs, at long time from IR (i.e., 24 h). Since some of the newly identified interactors of the p26 and p70 fragments have not been found to interact with the full-length NBN, these interactions may somehow contribute to the key biological phenomena underpinning NBS. Both nibrin and MRE11 are components of the PML-nuclear bodies (PML-NBs), nonmembrane-bound organelles within the nuclei of mammalian cells involved in several biological processes relevant to tumor suppression (e.g., induction of apoptosis, cell cycle checkpoints control, transcriptional regulation, posttranslational modification, chromatin remodeling, DDR, and DNA repair). PML-NBs increase in number and change their sub-nuclear distribution in response to DNA damage, and this response is dependent upon early DNA break-sensing proteins. Many proteins involved in DDR localize to PML-NBs either constitutively or conditionally, including the DNA damage-sensing proteins ATM, ATR, BRCA1, CHK2, p53, the MRN complex, and TopBP1, as well as multiple proteins that participate in DSBs repair. It is known that the physical integrity of PML-NBs is affected by DNA damaging agents like IR, thus leading to the hypothesis that PML-NBs may act as DNA damage sensors, their physical disruption representing a mechanism of DNA damage signaling. The disruption of PMLNBs consequent to the expression of the oncogenic PML-RARa fusion protein is responsible for acute promyelocytic leukemia (APL) onset. The aim of the third year of this PhD project was to study the relationship existing between PML-NBs integrity and IR-induced DSBs sensing, signaling and repair using both in vitro and in vivo models. Results obtained suggested that the expression of the PML-RARα fusion protein in APL and APL-like human cell lines (i.e., NB4 and U937/PR9 cells, respectively), in human primary cells freshly established from APL patients, as well as in a pre-blastic PML-RARα knock-in mouse model, causes a delayed sensing and signaling of the IR-induced DSBs. This result supports the hypothesis that a PML-NBs loss of integrity causes a dispersion of the proteins involved in the DDR, determining inability to respond quickly and correctly to DSBs. Remarkably, the pre-blastic PML-RARa knock-in model allowed us to study the effect of PML-RARα in the absence of any epigenetic and/or genetic alteration responsible for the clonal expansion typical of leukemic cells. In conclusion, results obtained highlighted the key role played by nibrin and PML in several phases of the DDR. Notably, alterations in DNA repair pathways that arise during tumor development can make some cancer cells reliant on a reduced set of DNA repair pathways for survival. There is evidence that drugs that inhibit one of these pathways in such tumors could prove useful as single-agent therapies, with the potential advantage that this approach could be selective for tumor cells and have fewer side effects. Hence, there is an urgent need to develop novel treatment strategies and this may be achieved by shading light on cancer biology in order to identify key pathways involved in the response to and repair of DNA damage.
Access Rights: info:eu-repo/semantics/openAccess
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