The many functions of APE1/Ref-1: not only a DNA repair enzyme

Abstract

APE1/Ref-1 (APE1), the mammalian ortholog of Escherichia coli Xth, and a multifunctional protein possessing both DNA repair and transcriptional regulatory activities, has a pleiotropic role in controlling cellular response to oxidative stress. APE1 is the main apurinic/apyrimidinic endonuclease in eukaryotic cells, playing a central role in the DNA base excision repair pathway of all DNA lesions (uracil, alkylated and oxidized, and abasic sites), including single-strand breaks, and has also cotranscriptional activity by modulating genes expression directly regulated by either ubiquitous (i.e., AP-1, Egr-1, NFkappa-B, p53, and HIF) and tissue specific (i.e., PEBP-2, Pax-5 and -8, and TTF-1) transcription factors. In addition, it controls the intracellular redox state by inhibiting the reactive oxygen species (ROS) production. At present, information is still inadequate regarding the molecular mechanisms responsible for the coordinated control of its several activities. Both expression and/or subcellular localization are altered in several metabolic and proliferative disorders such as in tumors and aging. Here, we have attempted to coalesce the most relevant information concerning APE1's different functions in order to shed new light and to focus current and future studies to fully understand this unique molecule that is acquiring more and more interest and translational relevance in the field of molecular medicine.

FIG. 1.

Representations of APE1 domains structure and functions. (A) Schematic structure, based on functional studies (66, 144) of APE1 structure with critical residues. Schematic diagram of putative or experimental determined post-translational modifications of APE1/Ref-1. CKI, casein kinase I; CKII, casein kinase II; GSK3, glycogen synthase kinase 3; NLS, nuclear localization signal; PKC, protein kinase C. (B) APE1 functions in base excision repair. FEN1, flap endonuclease 1; MPG, methylpurine DNA glycosylase; Ogg1, 8-oxoguanine DNA glycosylase, PCNA, proliferating cell nuclear antigen; RFC, replication factor C; XRCC1, X-ray cross-species complimenting 1. (C) Theoretical molecular model of the redox function of APE1 as a transcriptional coactivator of several transcription factors. Thioredoxin (Trx) is responsible for completing the redox cycle by which APE1 reduced form is restored. DBD, DNA binding domain.

FIG. 2.

Schematic representation of some of the stimuli known to activate APE1/Ref-1 expression and/or function. APE1 functional activation, which is associated to nuclear accumulation and upregulation of protein expression, is a consequence of different conditions and implies different outcomes. Thus, APE1 is a central actor in the adaptive cellular response to oxidative stress.

FIG. 3.

Different model networks for APE1-mediated signaling as derived by gene expression and proteomic data. Direct interaction network for genes dysregulated upon APE1 knock-down, involved in redox control, transcription regulators and chaperones (A) and (B) and in p53-signaling (C). Colors of the symbols indicate inhibition (blue) and activation (red). Here, the name APEX is used to refer to APE1. This model network is accessible at this site: https://portal.genego.com/pub/network/n-860173808.html. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 4.

Functional inactivation of Egr-1 transcriptional activity on PTEN target gene. HDACs inhibitors-induced PTEN upregulation requires APE1 expression. (A) Inducible expression of Egr-1 by 1 h serum treatment after o/n starvation is significantly reduced in APE1-silenced cells. Endogenous APE1 silencing was obtained by inducible RNAi strategy (30). Expression of Egr-1 and APE1 proteins was evaluated by Western blot analysis on 10 μg of nuclear extracts as previously described (30). (B) Induction of endogenous PTEN expression by HDACs inhibitors is inhibited in HeLa cells in which Egr-1 is diminished upon constitutive stable APE1 knock-down. To evaluate the endogenous PTEN levels upon HDACs inhibitors treatment, control and knocked-down HeLa cells were treated with TSA for 30 h and further collected and lysed to obtain total extracts that were subsequently used for Western blot analysis. Ten μg of extracts were separated onto a 10% SDS-PAGE, blotted onto nitrocellulose membranes, and assayed for the presence of APE1 protein by using the monoclonal anti-APE1 antibody, for the presence of PTEN protein by using the monoclonal anti-PTEN antibody and for the presence of Egr-1 protein by using a specific polyclonal antibody. Actin was also measured, as loading control. Values represent the relative amount of PTEN with respect to control untreated cells, after normalization for actin content. The mean value of two independent experiments is reported. (C) Induction of PTEN promoter activity by HDACs inhibitors is prevented in constitutive stable APE1 knocked-down HeLa cells in which Egr-1 is significantly diminished. APE1 silencing was obtained as described in legend to (A). After 10 days of doxycycline treatment, HeLa cells were transfected with plasmid containing the PTEN promoter-Luc sequence and then treated with TSA for 30 h. Twenty-four hours after treatment, cells were harvested, and luciferase and β-galactosidase activities were measured. Bars indicate the mean value ± SD of three independent experiments. (*p < 0.05 by Student's t-test). (D) Model network involving APE1-Egr 1 functional interaction. This model network is accessible at this site: https://portal.genego.com/pub/network/n-839812191.html. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 5.

(A) APE1 protein sequence bears several putative sumoylation sites, as from in silico analysis. APE1 protein contains 29 Lysine residues, 15 of them lie in the N-terminal domain of the protein. Protein sequence analysis using three different softwares for prediction of sumoylation sites (SUMOPlot, SUMO sp, and SUMO PSFS) revealed several “low probability” putative sumoylation sites and a “high probability” putative sumoylation site, which contains a canonical sumoylation ψ-K-X-D/E motif. Putative sumoylation sites do not distribute homogenously along whole sequence, but concentrate at N-terminal domain, which is not present in functionally related proteins from other organisms and is required for the redox activity of APE1. (B) Of the 29 Lys residues, 8 of them are identified as potential sumoylation sites with the three different softwares. Lys85represents a classical consensus sequence, recognized by all progr ams. Lys78 is recognized as low-probability consensus sites by two different programs, while five Lys residues (K3, K6, K27, K63, and K125) are recognized as potential sumoylation sites by only one program. Interestingly, all potential sumoylation sites reside within the Redox transactivation domain. (C) APE1 colocalizes with SUMO-1, but not with Δ6 SUMO-1, into nuclear subdomains, presumably nuclear bodies. HeLa cells were transfected with either pGFPSUMO-1 or pGFPΔ6SUMO-1, as a negative control. The inactive Δ6SUMO-1 is a deletionmutant lacking the 6 C-terminal aminocid residues, including double glycines being the site for the isopeptidic bond with the target protein, whichcannot sumoylate target proteins located in the nuclear bodies. Thus, this mutant relocalizes within nucleus and cytoplasm in a diffuse way. APE1 staining was analyzed using a monoclonal anti-APE1 antibody. Primary antibody and GFPSUMO-1 staining were revealed by incubation with Alexa Fluor 546 conjugated secondary antibody or by intrinsic green fluorescence of GFP, respectively. Merging of the two colors results in a yellow signal, corresponding to co-localized proteins. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 6.

Model of APE1 multifunctional roles in coordinating cell response to oxidative stress. APE1 has dual functions in cellular response tooxidative stress by acting as an AP-endonuclease in DNA repair and as a transcriptional regulator of various transcription factors leading to cell-cycle arrest or to cell survival. APE1's post-translational modifications and regulation of its intracellular trafficking may be critical in in vivo fine-tuning of its activities.