Cilli Domenica, Mirasole Cristiana, Pennisi Rosa, Pallotta Valeria, D'Alessandro Angelo, Antoccia Antonio, Zolla Lello, Ascenzi Paolo, di Masi Alessandra
Department of Science, Roma Tre University, Rome, Italy.
Department of Ecological and Biological Sciences, University of Tuscia, Viterbo, Italy.
PLoS One. 2014 Dec 8;9(12):e114651. doi: 10.1371/journal.pone.0114651. eCollection 2014.
Nibrin (also named NBN or NBS1) is a component of the MRE11/RAD50/NBN complex, which is involved in early steps of DNA double strand breaks sensing and repair. Mutations within the NBN gene are responsible for the Nijmegen breakage syndrome (NBS). The 90% of NBS patients are homozygous for the 657del5 mutation, which determines the synthesis of two truncated proteins of 26 kDa (p26) and 70 kDa (p70). Here, HEK293 cells have been exploited to transiently express either the full-length NBN protein or the p26 or p70 fragments, followed by affinity chromatography enrichment of the eluates. The application of an unsupervised proteomics approach, based upon SDS-PAGE separation and shotgun digestion of protein bands followed by MS/MS protein identification, indicates the occurrence of previously unreported protein interacting partners of the full-length NBN protein and the p26 fragment containing the FHA/BRCT1 domains, especially after cell irradiation. In particular, results obtained shed light on new possible roles of NBN and of the p26 fragment in ROS scavenging, in the DNA damage response, and in protein folding and degradation. In particular, here we show that p26 interacts with PARP1 after irradiation, 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 24 h from IR. 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.
尼布林（也称为NBN或NBS1）是MRE11/RAD50/NBN复合物的一个组成部分，该复合物参与DNA双链断裂感知和修复的早期步骤。NBN基因突变导致尼曼-匹克氏综合征（NBS）。90%的NBS患者是657del5突变的纯合子，该突变决定了两种截短蛋白的合成，即26 kDa（p26）和70 kDa（p70）。在这里，利用HEK293细胞瞬时表达全长NBN蛋白或p26或p70片段，然后通过亲和层析富集洗脱液。基于SDS-PAGE分离、蛋白质条带的鸟枪法消化以及随后的MS/MS蛋白质鉴定的无监督蛋白质组学方法的应用表明，全长NBN蛋白和含有FHA/BRCT1结构域的p26片段存在以前未报道的蛋白质相互作用伙伴，特别是在细胞照射后。特别是，所获得的结果揭示了NBN和p26片段在活性氧清除、DNA损伤反应以及蛋白质折叠和降解中的新的可能作用。特别是，我们在这里表明，p26在照射后与PARP1相互作用，并且这种相互作用对PARP1活性产生抑制作用，这通过NAD+水平来衡量。此外，p26-PARP1相互作用似乎是导致照射后24小时活性氧持续存在进而导致双链断裂持续存在的原因。由于尚未发现p26和p70片段的一些新鉴定的相互作用蛋白与全长NBN相互作用，这些相互作用可能以某种方式促成了支撑NBS的关键生物学现象。
