APE1 functional activation is a consequence of different stimuli that may generate both physiological and toxic oxidative stress conditions or increase the intracellular cAMP levels leading to different outcomes ().
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 (more ...)
The regulatory functions of the different APE1 activities can be fine-tuned and implemented via three different mechanisms: (a) increase in APE1's level after transcriptional activation (107
); (b) relocalization of APE1 from the cytoplasm to the nucleus (126
); and (c) modulation of APE1's post-translational modifications (PTM), such as acetylation and phosphorylation. As recently demonstrated, in addition to redox regulation, acetylation appears to have a fine-tuning role in affecting APE1's different activities (7
Both in vivo
and in vitro
studies demonstrated that different oxidative or toxic agents and/or intracellular produced ROS efficiently and rapidly (within minutes to hours, depending on the specific ROS-generating stimuli) promote a transient increase in APE1 protein levels, which is inhibited by cycloheximide (111
). Different transcription factors, including Sp-1 (36
), Egr-1 (107
), STAT3 (53
), CREB (48
), and Jun/ATF4 (37
) are involved in the inducible expression of APE1. APE1 itself may inhibit its own expression through the binding to nCa-RE sequences within the APE1 distal promoter, thus constituting an autoregulatory functional loop (68
). In addition, APE1 expression is linked in a positive autoregulatory loop with Egr-1 (107
), and in a negative inhibitory loop with p53 (151
). Interestingly, protein upregulation is always associated with an increase in both redox and AP endonuclease activity, followed by an increase in cell resistance toward oxidative stress and DNA damaging agents (49
), strengthening the conclusion that an upregulation of APE1 protein levels has profound biological consequences.
The activation of APE1 is also obtained by a process independent from de novo
synthesis and involves cytoplasm to nucleus translocation after exposure of cells to oxidative stress conditions (111
) or upon physiologic increase in intracellular ROS production (108
). Nuclear localization of APE1 is controlled by the first 20 amino acids at the N-terminal sequence, as determined by Jackson et al.
) and nuclear import is controlled through a bipartite NLS comprising residues 1–7 and 8–13 with the involvement of an importin system. In fact, the first 20 residues directly bind to karyopherin α1 and α2. Data obtained by treatment of cells with the nuclear export inhibitor leptomycin B suggested the presence of a nuclear export signal (NES) that may reside in a Leu-rich region (L291, L292, L295 residues, which are exposed in the 3-D structure) (69
). Recently, it has been shown that the region comprising amino acids 64–80 contains a NES (110
). Thus, both nuclear import and export may control subcellular distribution of APE1. In addition, the interaction with specific nuclear proteins could be a means to maintain APE1 within the nucleus. This hypothesis, recently proposed by Jung et al.
based on their data showing that nuclear localization of APE1 was dependent on GADD45a nuclear protein expression (71
), deserves further experimental support.
During the last few years, several lines of evidence have been accumulating, demonstrating that functional triggering of membrane-bound receptors (such as those for TSH, CD40L, ATP, IL-2, etc.
) can lead to APE1 functional activation through intracellular generation of sublethal doses of ROS (126
). Noteworthy is the observation that APE1 is also directly responsible for the control of the intracellular ROS levels through its inhibitory effect on Rac1 (2
, Vascotto et al.
, unpublished observations), the regulatory subunit of a membrane nonphagocytic NADPH oxidase system. This enzyme, composed of multiple membrane-associated (Mox and p22phox
) and cytosolic components (p67phox
, and Rac1), catalyzes the transformation of the molecular oxygen to the superoxide anion by transferring an electron from the substrates NADH or NADPH (3
). Since we have recently demonstrated that NADPH-mediated ROS production induced by P2Y triggering was able to promote APE1 functional activation (108
), we propose the existence of an autoregulatory loop between these two systems. This mechanism may be of therapeutic relevance for endothelial, fibroblastic, and smooth muscle cells, and should be analyzed in diseases of the vascular system where an overactivation of the NADPH oxidase system is involved (19
), as well as in the angiogenesis process (133
), where an additional autoregulatory loop between APE1 and VEGF may be inferred (10
). This observation could be therapeutically relevant in the treatment of tumor progression and cancer metastasis.
Based on the above-mentioned considerations, APE1 seems to act as an intracellular signaling tool involved both in modulating the cellular response to acute and chronic oxidative stress conditions, and also in controlling the endogenous ROS levels during the physiological generation of ROS as intracellular signaling molecules. Since the cell system must be able to discriminate between different ROS-generating stimuli, APE1 behaves as an integrating signaling molecule.
APE1 is an abundant protein (~104
copies/cell) within eukaryotic cells and with a relatively long half-life [~8
h (Vascotto et al.
, unpublished observations)]. Therefore, the fine-tuning of the multiple functions of this pleiotropic protein may reside in the impact that PTMs have on the function of APE1 and on the modulation of the APE1-interactome under different conditions. Whereas for the former hypothesis some experimental evidences have been obtained [acetylation of K6/K7 residues (7
)], very little information is now available on the protein interacting partners of APE1. Pioneering in silico
studies discovered that several different phosphorylation sites were scattered throughout the molecule. These potential phosphorylation sites included consensus sequences for casein kinase I and II (CKI and CKII), for protein kinase C (PKC), and for GSK3 () (34
). Initial in vivo
studies confirmed a role for PKC in phosphorylating APE1 in response to PMA or to alkylating agents (i.e.
, MMS) leading to AP-1 activation (62
). However, these studies have not been repeated nor followed up. Therefore, the role of phosphorylation on APE1 is still not clear.
APE1 is a site for redox regulation by the dithiol-reducing enzyme Trx (61
), through Cys35 and Cys32 in the catalytic center of Trx, and involving the Cys65 redox sensitive site of APE1 (139
). The Trx-mediated redox regulation of APE1 is required for the functional activation of p53 (132
) and AP-1 (61
). Though the biological relevance of Cys65 residue seems determined, it is not currently known whether this Cys residue undergoes PTM in vivo.
Qu et al.
) demonstrated that two (Cys93 and Cys310) of the seven Cys residues of APE1 can undergo S
-nitrosation in response to nitric oxide stimulation, leading to nucleus to cytoplasm relocalization of the protein in a CRM1-independent process, possibly as a consequence of demasking a putative nuclear export signal (aa 64–80). S
-nitrosation may therefore constitute a specific molecular switch to strictly control the intracellular distribution of APE1 between nucleus and cytoplasm, and provides a new working hypothesis for the cytoplasmic accumulation of APE1 observed in more aggressive tumors (126
). Unfortunately, no detail is available about the functional implications of S
-nitrosation on the different biological functions of APE1. Accordingly, since both NO and APE1 are associated with tumorigenesis and neurodegenerative diseases, future work is needed to address whether nitrosative stress leads to genomic instability, and may be the target for designing new therapeutic strategies.
An interesting post-translational processing that has been recently described is proteolysis occurring at residue Lys31. This PT regulation of APE1 protein is responsible for enhanced cell death mediated by granzyme A (GzmA) (29
) and granzyme K (GzmK) (52
). APE1 is associated with the endoplasmic reticulum in a macromolecular complex of 270–420
kDa containing evolutionarily conserved proteins called SET, pp32, and HMG2. GzmA cleaves APE1 after Lys31, giving rise to a protein form called NΔ33APE1, and alters its ability to be actively accumulated within nuclei of cells (15
, and our unpublished observations) and to interact with XRCC1 (136
). However, some authors claimed that truncated APE1 may loose its AP-endonuclease activity (29
) and acquire a nonspecific DNAse function (150
). This peculiar processing is not limited to immune cells but may constitute a general molecular device for redirecting APE1 to mitochondria (125
), as suggested by Chattopadhyay et al.
) and Mitra et al.
), in spite of the intriguing finding of a proteolysis occurring at the level of Asn33 rather than Lys31. Again, if the removal of the terminal 31–33 amino acids is responsible for APE1 to move to the mitochondria to function in mitochondrial BER as an AP endonuclease, it is hard to understand this truncated protein having nonspecific DNAse activity (150
) unless it is very cell-type specific. Accordingly, previous work by many investigators has never observed a nonspecific nuclease activity with the cleavage of the first 61 amino acids (66
) and additional data clearly showed that the truncated APE1 protein has an unaltered AP-endonuclease activity (15
, and our unpublished observations), at least in vitro.
While it is known that nuclear accumulation of APE1 triggers the activation of several transcription factors, the functional role of acetylation is barely understood. Acetylation of both histones and regulatory proteins is commonly catalyzed by the histone acetyltransferase (HAT) p300/CBP, and can be reversed by histone deacetylases (HDACs), which in turn control the acetylation level of transcription factors or co-activators (50
). Bhakat et al.
have reported that the balance between the acetyltransferase activity of p300/CBP and the deacetylase activity of HDAC1 maintains APE1's acetylation at Lys residues 6 and 7 (K6, K7) in response to Ca2+
levels, thus controlling expression of target genes (7
). More recently, we found that exposure of HeLa cells to H2
and to histone deacetylase inhibitors increases acetylation of APE1 at residues Lys6/Lys7, leading to Egr-1-mediated induction of the tumor suppressor PTEN gene expression (30
). Our data open new perspectives in the comprehension of the many functions exerted by APE1 in controlling cell response to oxidative stress and underline the double-face nature of APE1 which plays a role in both pro-survival and in cell cycle arrest mechanisms. Interestingly, despite the very low homology degree in the N-terminal region (<40%), K6 or K6/K7 are much more conserved, thus reinforcing their primary role during phylogenesis.
Altogether, these observations have raised the possibility that subtle PTMs provide a means for channeling the multifunctional APE1 to different activities and interactions and thus could act as a regulatory switch in performing different functions. APE1 subcellular localization is quite variable. Most cell types exhibit only nuclear, others display only cytoplasmic, while others show both nuclear and cytoplasmic localization (126
). Such a complex distribution pattern suggests that localization is not random but, on the contrary, is controlled by a strictly regulated process. Though of fundamental interest for a full comprehension of the role of APE1 in different pathological conditions, the clear understanding of the biological relevance of APE1 subcellular compartmentalization still remains elusive. Whereas we can rather easily figure out the role for nuclear localization of APE1 based on its main DNA repair and co-transcriptional activity, a convincing explanation for the extranuclear roles of APE1 is still evanescent. Cytoplasmic localization of APE1, such as that reported for fibroblasts, spermatocytes, thyrocytes, lymphocytes, hepatocytes, and hippocampal cells (20
), is associated with high metabolic or proliferative rates and may be related to a cell cycle-dependent expression (36
). Possible explanatory hypotheses for cytoplasmic expression of APE1 may come from the mitochondrial role of the protein, as described above. A further functional explanation comes from its association with endoplasmic reticulum membranes, as evidenced by ultrastructural (125
) and biochemical (28
) analysis. It has been suggested that APE1 redox activity in the cytoplasm may be required to maintain newly synthesized transcription factors in a reduced state during their translocation to the nucleus (24
). Therefore, future work is required to shed more light on the extranuclear role(s) of APE1, starting from the explanation of its cytoplasmic function.