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 ...

Apurinic/apyrimidinic endonuclease 1 (APE1), an essential protein in mammals, is involved in base excision DNA repair (BER) and in regulation of gene expression, acting as a redox co-activator of several transcription factors. Recent findings highlight a novel role for APE1 in RNA metabolism, which is modulated by nucleophosmin (NPM1). The results reported in this article show that five lysine residues (K24, K25, K27, K31 and K32), located in the APE1 N-terminal unstructured domain, are involved in the interaction of APE1 with both RNA and NPM1, thus supporting a competitive binding mechanism. Data from kinetic experiments demonstrate that the APE1 N-terminal domain also serves as a device for fine regulation of protein catalytic activity on abasic DNA. Interestingly, some of these critical lysine residues undergo acetylation in vivo. These results suggest that protein–protein interactions and/or post-translational modifications involving APE1 N-terminal domain may play important in vivo roles, in better coordinating and fine-tuning protein BER activity and function on RNA metabolism.

APE1/Ref-1 protects cells from oxidative stress by acting as a central enzyme in base excision repair pathways of DNA lesions and through its independent activity as a redox transcriptional co-activator. Dysregulation of this protein has been associated to cancer development. At present, contrasting data have been published regarding the biological relevance of the two functions as well as the molecular mechanisms involved. Here, we combined both mRNA expression profiling and proteomic analysis to determine the molecular changes associated with APE1 loss-of-expression induced by siRNA technology. This approach identified a role of APE1 in cell growth, apoptosis, intracellular redox state, mitochondrial function and cytoskeletal structure. Thus, overall, our data show that APE1 acts as a hub in coordinating different and vital functions in mammalian cells, highlighting the molecular determinants of the multifunctional nature of APE1 protein.

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