鉴定人神经纤维蛋白（NBN）的相互作用因子及其由NBN 657del5奠基者突变产生的26 kDa和70 kDa片段。,PLOS ONE

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鉴定人神经纤维蛋白（NBN）的相互作用因子及其由NBN 657del5奠基者突变产生的26 kDa和70 kDa片段。
PLOS ONE ( IF 2.9 ) Pub Date : 2014-12-09 , DOI: 10.1371/journal.pone.0114651
Domenica Cilli ₁ , Cristiana Mirasole ₂ , Rosa Pennisi ₁ , Valeria Pallotta ₂ , Angelo D'Alessandro ₂ , Antonio Antoccia ₃ , Lello Zolla ₂ , Paolo Ascenzi ₄ , Alessandra di Masi ₃

Affiliation

Nibrin（也称为NBN或NBS1）是MRE11 / RAD50 / NBN复合物的组成部分，它参与DNA双链断裂感测和修复的早期步骤。NBN基因内的突变是奈梅亨断裂综合症（NBS）的原因。90％的NBS患者是657del5突变的纯合子，该突变决定了26 kDa（p26）和70 kDa（p70）两种截短蛋白的合成。在这里，已经利用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。一种无监督蛋白质组学方法的应用，该方法基于SDS-PAGE分离和shot弹枪消化蛋白条带，然后进行MS / MS蛋白质鉴定，表示全长NBN蛋白和含有FHA / BRCT1域的p26片段以前未报告的蛋白相互作用伴侣的出现，尤其是在细胞照射后。特别是，获得的结果揭示了NBN和p26片段在ROS清除，DNA损伤反应以及蛋白质折叠和降解中的新可能作用。特别是，在这里我们显示p26在辐照后与PARP1相互作用，并且该相互作用对PARP1的活性产生抑制作用（通过NAD +水平测量）。此外，p26-PARP1相互作用似乎是IR持续24小时后ROS以及DSB持续存在的原因。由于尚未发现一些新鉴定的p26和p70片段相互作用子与全长NBN相互作用，

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Identification of the interactors of human nibrin (NBN) and of its 26 kDa and 70 kDa fragments arising from the NBN 657del5 founder mutation.

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.

更新日期：2019-11-01

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