Evolution of the redox function in mammalian apurinic/apyrimidinic endonuclease

Abstract

Human apurinic/apyrimidinic endonuclease (hApe1) encodes two important functional activities: an essential base excision repair (BER) activity and a redox activity that regulates expression of a number of genes through reduction of their transcription factors, AP-1, NFκB, HIF-1α, CREB, p53 and others. The BER function is highly conserved from prokaryotes (E. coli exonuclease III) to humans (hApe1). Here, we provide evidence supporting a redox function unique to mammalian Apes. An evolutionary analysis of Ape sequences reveals that, of the 7 Cys residues, Cys 93, 99, 208, 296, and 310 are conserved in both mammalian and non-mammalian vertebrate Apes, while Cys 65 is unique to mammalian Apes. In the zebrafish Ape (zApe), selected as the vertebrate sequence most distant from human, the residue equivalent to Cys 65 is Thr 58. The wild-type zApe enzyme was tested for redox activity in both in vitro EMSA and transactivation assays and found to be inactive, similar to C65A hApe1. Substitution of Thr 58 with Cys in zApe, however, resulted in a redox active enzyme, suggesting that a Cys residue in this position is indeed critical for redox function. In order to further probe differences between redox active and inactive enzymes, we have determined the crystal structures of vertebrate redox inactive enzymes, the C65A human Ape1 enzyme and the zApe enzyme at 1.9 and 2.3 Å, respectively. Our results provide new insights on the redox function and highlight a dramatic gain-of-function activity for Ape1 in mammals not found in non-mammalian vertebrates or lower organisms.

Introduction

Redox regulation has been shown to play an important role in modulating the DNA binding activity of a number of transcription factors including AP-1, NFκB, HIF-1α, HLF, CREB, PAX, p53 and others [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11] (reviewed in [12]). As described for AP-1 (Fos/Jun), this regulation involves the reduction of a critical Cys residue(s) within c-Jun that allows it to bind DNA with much higher affinity than the oxidized c-Jun [13], [14]. The factor responsible for reducing AP-1 was found to be a nuclear factor, termed redox effector factor 1 (Ref1) [15], and later identified as apurinic/apyrimidinic endonuclease (Ape1, also referred to as HAP1 or APEX1) [2]. Ape1 is an essential enzyme in the base excision repair pathway catalyzing the second step in the pathway, cleavage of the phosphodiester backbone 5′ of the abasic site leaving a 3′ hydroxyl and 5′-deoxyribose phosphate. Deletion of the APE1 gene results in very early embryonic lethality in mice [16], and knock-down of Ape through the use of a morpholino oligonucleotide injected in zebrafish embryos results in death at the midblastula transition [17]. In addition, Ape has been shown to be involved in heart and blood development in zebrafish [17].

The redox activity has been shown to require the N-terminal region of the protein (reviewed in [12]). The human Ape1 includes 61 residues not present in exonuclease III from E. coli, and while truncations of up to 40 residues do not affect the redox activity, removal of the 62 N-terminal residues from the human Ape1 renders this enzyme redox inactive [14], [18]. It was hypothesized that Ape1's redox activity might involve a Cys residue [14]. Accordingly, each of the seven Cys residues in Ape1 was substituted individually with Ala, and the resulting enzymes were tested for their ability to reduce AP-1 thereby allowing it to bind target DNA specifically in an electrophoretic mobility shift assay (EMSA) [18]. Of these single Cys mutants, all when reduced retained wild-type redox activity except C65A hApe1, which was redox inactive. When oxidized, C93A hApe1 was found to be redox active, while all of the other mutants were inactive. Thus, a redox mechanism involving a disulfide bond between Cys 65 and Cys 93 was proposed [18].

Subsequently, however, several crystal structures of Ape1 have been reported [19], [20], [21] providing three important observations with regard to the redox activity of Ape1. First, no disulfide bonds are present in any of the structures. Second, Cys 65 is a buried residue in the conformation of hApe1 observed in the crystal structures and therefore not accessible to the transcription factors that Ape1 reduces. Third, the structures of hApe1 that have been reported to date in different crystallographic lattices are remarkably similar. Even in the hApe1–DNA complex, there are no significant conformational changes in the enzyme [20]. Thus, the mechanism by which Cys 65 would participate in a potential redox reaction between Ape1 and a transcription factor requires further investigation. One possible explanation offered by Gorman et al. [19] was that substitution of Cys 65 with Ala may affect the stability and/or folding of Ape1 and result in subsequent loss of the redox activity. More recently, the role of Cys 65 in Ape1's redox activity has been challenged through the creation of a Ref1^C64A/C64A mouse (C64 is equivalent to the human C65 residue) that was shown to develop normally [22]; in this study the authors concluded that Cys 65 is not required for redox activity.

Ape vertebrate sequences are highly conserved with the most distantly related sequence, that of zebrafish Ape (zApe) being 66% identical to the human Ape1. We had initially selected zebrafish as a potential model system to study the role of Ape in development as mice knockouts were lethal. However, as reported here, the wild-type zApe lacks redox activity. Thus, we have not pursued the zebrafish development experiments. We report the results of biochemical studies including hApe1, zApe, and a zApe mutant engineered to acquire redox function, the first crystal structures of redox inactive enzymes from vertebrates, and a discussion of the mechanistic implications for the redox activity of Ape.