Identification of the Interactors of Human Nibrin (NBN) and of Its 26 kDa and 70 kDa Fragments Arising from the NBN 657del5 Founder Mutation

Abstract

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.

DNA repair functional analyses of NBN hypomorphic variants associated with NBN‐related infertility

“…The N‐terminal part is composed of one FHA domain (aa 24–108) and two BRCT (aa 108–196 and aa 218–317). This N‐terminal region allows interaction with numerous DNA‐damage response proteins, including PARP1, MDC1, and CtIP, essentially after phosphorylation or PARylation (Cilli et al, ; Li et al, ; Lloyd et al, ; Williams et al, ). The interaction domain for MRE11 is located in the NBN C‐terminal region (aa 640–691) and the interaction domain for ATM on the last 20 amino acids of the protein (aa 734–754; Dupre et al, ; Schiller et al, ).…”

“…It has been shown that p26‐NBN transiently expressed could interact with CtIP. However, p26‐NBN does not contain the MRE11‐interaction domain and probably cannot mediate the recruitment of the MRN complex and the activation of the endonuclease activity of MRE11 (Cilli et al, ; Li et al, ; Lloyd et al, ; Williams et al, ). Despite the expected presence of the FHA domain in the patient's NBN variant, Varon's patient had microcephaly, although not as severe as typical patients with NBS.…”

The Slavic NBN Founder Mutation: A Role for Reproductive Fitness?

“…Carriers of the NBN c.657del5 mutation have a distinct gene expression phenotype with about the same number of genes up- and down regulated [67]. Moreover, based upon SDS-PAGE and mass spectrometry, it was shown that the two truncated proteins, p26 and p70, synthesized by carriers of the mutation, bind to proteins that do not interact with full-length nibrin [68]. It is tempting to speculate that these effects are also relevant in maternal germ cells and could explain the increase in fecundity of carriers.…”

Hsp90α regulates ATM and NBN functions in sensing and repair of DNA double‐strand breaks

“…Previous data from our lab demonstrated that Hsp90α and NBN interact both in unstressed condition and following IR‐induced damage in HEK293 cells . To clarify the role of Hsp90α in the interaction between ATM and NBN during the DDR, co‐immunoprecipitation (IP) experiments were performed in cells exposed to 4 Gy of X‐rays and harvested after 0.5, 3, and 6 h.…”