APE1/Ref-1 Interacts with NPM1 within Nucleoli and Plays a Role in the rRNA Quality Control Process

Abstract

APE1/Ref-1 (hereafter, APE1), a DNA repair enzyme and a transcriptional coactivator, is a vital protein in mammals. Its role in controlling cell growth and the molecular mechanisms that fine-tune its different cellular functions are still not known. By an unbiased proteomic approach, we have identified and characterized several novel APE1 partners which, unexpectedly, include a number of proteins involved in ribosome biogenesis and RNA processing. In particular, a novel interaction between nucleophosmin (NPM1) and APE1 was characterized. We observed that the 33 N-terminal residues of APE1 are required for stable interaction with the NPM1 oligomerization domain. As a consequence of the interaction with NPM1 and RNA, APE1 is localized within the nucleolus and this localization depends on cell cycle and active rRNA transcription. NPM1 stimulates APE1 endonuclease activity on abasic double-stranded DNA (dsDNA) but decreases APE1 endonuclease activity on abasic single-stranded RNA (ssRNA) by masking the N-terminal region of APE1 required for stable RNA binding. In APE1-knocked-down cells, pre-rRNA synthesis and rRNA processing were not affected but inability to remove 8-hydroxyguanine-containing rRNA upon oxidative stress, impaired translation, lower intracellular protein content, and decreased cell growth rate were found. Our data demonstrate that APE1 affects cell growth by directly acting on RNA quality control mechanisms, thus affecting gene expression through posttranscriptional mechanisms.APE1/Ref-1 (also called HAP1 or APEX, and here referred to as APE1), the mammalian ortholog of Escherichia coli Xth (exonuclease III), is a vital protein (20) that acts as a master regulator of cellular response to oxidative stress conditions and contributes to the maintenance of genome stability (55, 56). APE1 is involved in both the base excision repair (BER) pathways of DNA lesions, acting as the major apurinic/apyrimidinic (AP) endonuclease, and in transcriptional regulation of gene expression as a redox coactivator of different transcription factors, such as early growth response protein 1 (Egr-1), NF-B, and p53 (55, 56). These two biological activities are located in two functionally distinct protein domains. In fact, the N-terminal region, containing the nuclear localization signal (NLS) sequence, is principally devoted to redox activity through Cys65, while the C-terminal one exerts enzymatic activity on the abasic sites of DNA (56, 63). The protein C terminus is highly conserved during phylogenesis, while the N terminus is not. Except in mammals, which always show a high sequence conservation (more than 90%), and Danio, Drosophila, Xenopus, and Dictyostelium (presenting a sequence identity of less than 40%), the N-terminal region is mostly absent in other organisms. A third APE1 function, which is regulated by Lys6/Lys7 acetylation (7), is indirect binding to the negative calcium response elements (nCaRE) of some promoters (i.e., PTH and APE1 promoters), thus acting as a transcriptional repressor (12,30).In different ...

Nucleolin and nucleophosmin: nucleolar proteins with multiple functions in DNA repair

“…Thirdly, under the model outlined above, NPM1 is expected to act as a negative regulator of APE1. However, studies have instead observed that, in vitro, NPM1 binding to APE1 enhances its AP-endonuclease activity (Vascotto et al 2009) lysates, despite no longer being sequestered in nucleoli (Vascotto et al 2014). NPM1 -/-cells also showed an increased sensitivity to BER-inducing agents such as H 2 O 2 , KBrO 3 and MMS, and carried a greater number of AP-sites compared to NPM1 +/+ cells, suggesting that NPM1 is in fact required for APE1 function in vivo (Vascotto et al 2014).…”

“…The first is that, while APE1 is enriched in the nucleoli, extensive nucleoplasmic APE1 staining is still observed in most cells, suggesting that NPM1 sequesters only a relatively minor pool (an estimated 5-10% of total cellular APE1) into nucleoli (Lirussi et al 2012;Vascotto et al 2009). …”

“…This interaction, mediated by binding of the lysine-rich APE1 N-terminal tail to NPM1's N-terminal region containing the oligomerization domain and acidic tracts, results in the robust recruitment of APE1 into nucleoli (Fantini et al 2010;Vascotto et al 2009). Upon induction of genotoxic stress such as base D r a f t 23 oxidation or methylation, lysines within the APE1 N-terminal become acetylated, interrupting the NPM1-APE1 interactions and inducing translocation of APE1 from the nucleolus to the nucleoplasm (Fantini et al 2010;Lirussi et al 2012).…”

“…The first evidence for an involvement of NPM1 in the regulation of BER came from proteomic studies that identified NPM1 as a major interactor of APE1 in unstressed cells (Vascotto et al 2009). This interaction, mediated by binding of the lysine-rich APE1 N-terminal tail to NPM1's N-terminal region containing the oligomerization domain and acidic tracts, results in the robust recruitment of APE1 into nucleoli (Fantini et al 2010;Vascotto et al 2009).…”

Alterations in the expression of the apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) in human ovarian cancer and indentification of the therapeutic potential of APE1/Ref-1 inhibitor

“…Previous flow cytometric studies have indicated that the effect of APE1 siRNA on cisplatin sensitivity may be partly due to the modulation of the cell cycle, as a result of cellular response to DNA damage. New data demonstrate that APE1 affects cell growth by directly acting on RNA quality control mechanisms, thus affecting gene expression through post-transcriptional mechanisms (32). Future research is necessary in this area to identify the relationship between APE1 and cell cycle.…”