Reactive oxygen species (ROS) are generated as by-products of normal cellular metabolic activities. Superoxide dismutase, glutathione peroxidase, and catalase are the enzymes involved in protecting cells from the damaging effects of ROS. ROS are produced in response to ultraviolet radiation, cigarette smoking, alcohol, nonsteroidal anti-inflammatory drugs, ischemia-reperfusion injury, chronic infections, and inflammatory disorders. Disruption of normal cellular homeostasis by redox signaling may result in cardiovascular, neurodegenerative diseases and cancer. ROS are produced within the gastrointestinal (GI) tract, but their roles in pathophysiology and disease pathogenesis have not been well studied. Despite the protective barrier provided by the mucosa, ingested materials and microbial pathogens can induce oxidative injury and GI inflammatory responses involving the epithelium and immune/inflammatory cells. The pathogenesis of various GI diseases including peptic ulcers, gastrointestinal cancers, and inflammatory bowel disease is in part due to oxidative stress. Unraveling the signaling events initiated at the cellular level by oxidative free radicals as well as the physiological responses to such stress is important to better understand disease pathogenesis and to develop new therapies to manage a variety of conditions for which current therapies are not always sufficient.

Abasic (AP)-endonuclease (APE) is responsible for repair of AP sites, and single-strand DNA breaks with 3′ blocking groups that are generated either spontaneously or during repair of damaged or abnormal bases via the DNA base excision repair (BER) pathway in both nucleus and mitochondria. Mammalian cells express only one nuclear APE, 36 kDa APE1, which is essential for survival. Mammalian mitochondrial (mt) BER enzymes other than mtAPE have been characterized. In order to identify and characterize mtAPE, we purified the APE activity from beef liver mitochondria to near homogeneity, and showed that the mtAPE which has 3-fold higher specific activity relative to APE1 is derived from the latter with deletion of 33 N-terminal residues which contain the nuclear localization signal. The mtAPE-sized product could be generated by incubating 35S-labeled APE1 with crude mitochondrial extract, but not with cytosolic or nuclear extract, suggesting that cleavage of APE1 by a specific mitochondria-associated N-terminal peptidase is a prerequisite for mitochondrial import. The low abundance of mtAPE, particularly in cultured cells might be the reason for its earlier lack of detection by western analysis.

The mammalian AP-endonuclease APE1 is a ubiquitous and remarkably multifunctional protein. It plays a central role in the base excision repair (BER) pathway for repairing damaged bases and DNA single-strand breaks induced by reactive oxygen species and alkylating agents and also repairing AP sites that are generated spontaneously or after the excision of oxidized and alkylated bases by DNA glycosylases (12,40). APE1 was also shown to incise DNA 5Ј to oxidatively damaged bases including 5,6-dihydrothymidine and alpha-2Ј-deoxyadenosine in the nucleotide incision repair pathway (11,21). The deletion of the N-terminal 61 amino acid residues, which are dispensable for the AP-endonuclease activity, affected its incision activity as well (21, 28). Moreover, the ability of APE1 to incise DNA 5Ј to a bulky exocyclic adduct such as p-benozoquinone has also been reported (22). Besides its repair function, mammalian APE1 has two unique and apparently distinct transcriptional regulatory activities. It was independently identified as a reductive activator of c-Jun in vitro and named . Subsequently, several other transcription factors (including p53, NF-B, hypoxia-inducible factor 1-␣, PAX5, and PAX8) were also shown to be activated by APE1, presumably via the same redox process (13,31,55). A third and distinct function of APE1 as a trans-acting factor was discovered when APE1 was identified as being one of the regulatory proteins that binds to the negative Ca 2ϩ response elements (nCaRE-A and -B) in the Ca 2ϩ -dependent downregulation of the parathyroid hormone gene (48) and subsequently in the human renin gene (17). We later showed that human APE1 is acetylated at Lys6 and Lys7 by the histone acetyltransferase p300, both in vivo and in vitro, and that acetylation enhances APE1's binding to nCaRE-B, leading to the repression of the parathyroid hormone promoter (2). Our follow-up studies indicated that a small but significant fraction of APE1 in HeLa cells and mouse liver is present in the acetylated form (14,56). Recently, we have shown that the early growth response protein (Egr-1)-mediated activation of phosphoinositol phosphatase and tensin homologue (PTEN) is dependent on APE1 acetylation (14).Although APE1 heterozygous mice are viable and appear to be normal, APE1 nullizygous mice show early embryonic lethality (37,39,63). We recently showed that APE1 inactivation induced apoptosis in mouse embryo fibroblasts conditionally nullizygous for endogenous APE1, an effect that could be prevented by the ectopic expression of human APE1 (27). Using a complementation assay, we also showed that both the repair activity and acetylation-mediated transcriptional regulatory functions of APE1 are required to prevent the apoptosis of APE1-null mouse embryo fibroblasts. The unexpected essentiality of APE1's regulatory function suggests that APE1 is a coregulator of many critical genes.Resistance to many common anticancer drugs often occurs due to the enhanced expression of ATP-binding cassette (ABC) transporter proteins (6,20). Among the ...

The recently characterized enzyme NEIL2 (Nei-like-2), one of the four oxidized base-specific DNA glycosylases (OGG1, NTH1, NEIL1, and NEIL2) in mammalian cells, has poor base excision activity from duplex DNA. To test the possibility that one or more proteins modulate its activity in vivo, we performed mass spectrometric analysis of the NEIL2 immunocomplex and identified Y box-binding (YB-1) protein as a stably interacting partner of NEIL2. We show here that YB-1 not only interacts physically with NEIL2, but it also cooperates functionally by stimulating its base excision activity by 7-fold. Moreover, YB-1 interacts with the other NEIL2-associated BER proteins, namely, DNA ligase III␣ and DNA polymerase ␤ and thus could form a large multiprotein complex. YB-1, normally present in the cytoplasm, translocates to the nucleus during UVA-induced oxidative stress, concomitant with its increased association with and activation of NEIL2. NEIL2-initiated base excision activity is significantly reduced in YB-1-depleted cells. YB-1 thus appears to have a novel regulatory role in NEIL2-mediated repair under oxidative stress. Reactive oxygen species (ROS)2 are believed to play an important role in inducing various disease pathologies including cancer, rheumatoid arthritis, cardiovascular disease, and aging (1, 2). ROS are formed endogenously as a byproduct of respiration and oxidative metabolism and exogenously by a variety of environmental agents including ultraviolet (UV) radiation (3-5). Of the total ultraviolet light spectrum (100 -400 nm) present in the natural sunlight, only UVA (320 -400 nm) and a small fraction of UVB (280 -320 nm) reach the surface of the earth, as UVB is mostly absorbed by the atmosphere. Although UVA is the predominant genotoxic radiation in the natural environment, it does not react directly with DNA, but interacts with cellular chromophores like riboflavin, porphyrins, quinones, and reduced nicotinamide cofactors to produce singlet oxygen (6). UVA also has the ability to penetrate deeper into the skin to proliferating basal layers, and causes DNA damage leading to genomic instability.Although cellular antioxidant defenses (e.g. catalase, peroxidase, and superoxide dismutase) effectively combat the effects of ROS, but oxidative DNA damage still occurs. Most of the DNA lesions, except double strand breaks, are repaired via the DNA base excision repair (BER) pathway, initiated with the excision of damaged base by a specific DNA glycosylase (7,8). Four oxidized base-specific DNA glycosylases have been identified and characterized so far in mammalian cells. 8-Oxoguanine-DNA glycosylase (OGG1) and endonuclease III homolog 1 (NTH1) were characterized previously and preferentially excise oxidized purines and pyrimidines, respectively (9, 10), and were thought to be the two major oxidized base-specific DNA glycosylases in mammalian cells. However, a lack of phenotype or significant cancer propensity of OGG1-and NTH1-null mice suggested the contribution of other DNA glycosylases in the repair of oxidized ...

The mammalian abasic-endonuclease1/redox-factor1 (APE1/Ref1) is an essential protein whose subcellular distribution depends on the cellular physiological status. However, its nuclear localization signals have not been studied in detail. We examined nuclear translocation of APE1, by monitoring enhanced green fluorescent protein (EGFP) fused to APE1. APE1's nuclear localization was significantly decreased by deleting 20 amino acid residues from its N-terminus. Fusion of APE1's N-terminal 20 residues directed nuclear localization of EGFP. An APE1 mutant lacking the seven N-terminal residues (ND7 APE1) showed nearly normal nuclear localization, which was drastically reduced when the deletion was combined with the E12A/D13A double mutation. On the other hand, nearly normal nuclear localization of the full-length E12A/D13A mutant suggests that the first 7 residues and residues 8–13 can independently promote nuclear import. Both far-western analyses and immuno-pull-down assays indicate interaction of APE1 with karyopherin alpha 1 and 2, which requires the 20 N-terminal residues and implicates nuclear importins in APE1's nuclear translocation. Nuclear accumulation of the ND7 APE1(E12A/D13A) mutant after treatment with the nuclear export inhibitor leptomycin B suggests the presence of a previously unidentified nuclear export signal, and the subcellular distribution of APE1 may be regulated by both nuclear import and export.