Identification and Characterization of Mitochondrial Targeting Sequence of Human Apurinic/Apyrimidinic Endonuclease 1

Abstract

Dually targeted mitochondrial proteins usually possess an unconventional mitochondrial targeting sequence (MTS), which makes them difficult to predict by current bioinformatics approaches. Human apurinic/apyrimidinic endonuclease (APE1) plays a central role in the cellular response to oxidative stress. It is a dually targeted protein preferentially residing in the nucleus with conditional distribution in the mitochondria. However, the mitochondrial translocation mechanism of APE1 is not well characterized because it harbors an unconventional MTS that is difficult to predict by bioinformatics analysis. Two experimental approaches were combined in this study to identify the MTS of APE1. First, the interactions between the peptides from APE1 and the three purified translocase receptors of the outer mitochondrial membrane (Tom) were evaluated using a peptide array screen. Consequently, the intracellular distribution of green fluorescent protein-tagged, truncated, or mutated APE1 proteins was traced by tag detection. The results demonstrated that the only MTS of APE1 is harbored within residues 289 -318 in the C terminus, which is normally masked by the intact N-terminal structure. As a dually targeted mitochondrial protein, APE1 possesses a special distribution pattern of different subcellular targeting signals, the identification of which sheds light on future prediction of MTSs.Human APE1 (apurinic/apyrimidinic endonuclease) is an important multifunctional protein that plays a central role in the cellular response to oxidative stress. The two major activities of APE1 are DNA repair and redox regulation of transcriptional factors. On one hand, APE1 functions as a critical ratelimiting enzyme in DNA base excision repair and accounts for nearly all of the AP site incision activities in cell extracts (1). On the other hand, APE1 also exerts unique redox activity to regulate the DNA binding affinity of certain transcriptional factors by controlling the redox status of their DNA-binding domain (2). Inhibition of the redox function of APE1 blocks murine endothelial cell growth and angiogenesis and also blocks the growth of human tumor cell lines (3). The biological importance of APE1 is highlighted by the finding that APE1 knockout mice exhibit an embryonic lethal phenotype (4). Although APE1 has long been labeled as a nuclear protein, a growing body of evidence has shown that the subcellular distribution of APE1 can be cytoplasmic in some cell types with high metabolic or proliferative rates, with predominant localization in the mitochondria and the endoplasmic reticulum (5-7). Recent mitochondrial proteomic studies have further confirmed the existence of APE1 in the mitochondria (8). Considering the importance of the mitochondria in cellular response to oxidative stress, the roles of APE1 in the mitochondria have been extensively investigated.

Role of tyrosyl-DNA phosphodiesterase (TDP1) in mitochondria

“…For instance, 8-oxoguanine-DNA glycosylase (OGG1) has an associated lyase activity that breaks the phosphodiester backbone 3′ to the abasic site with generation of 5′-phosphate and 3′-blocking groups (15). Until now, APE1 was the only known 3′-processing repair enzyme in mitochondria (14,28). However, the 3′-exonuclease/phosphodiesterase activity of APE1 is relatively weak (26) and its tyrosyl-DNA phosphodiesterase activity is even weaker (34).…”

“…2A) and does not contain a MTS sequence (10,60). This implies that TDP1, like many other proteins and transcription factors such as APE1, FEN1, NF-kB, p53, and BRCA1 (23) that act both in the nucleus and mitochondria, enter mitochondria despite their lack of canonical mitochondria targeting sequences, indicating the existence of still unknown mechanisms of intracellular trafficking (13,14,28,61,62).…”

“…Abasic endonuclease 1 (APE1) is the a well characterized enzyme in mammals for the repair of 3′-phosphoglycolate esters (3′-PG) during oxidative DNA damage (25)(26)(27). Recently, APE1 has been shown to be localized both in nuclei and mitochondria (14,28). However, the 3′-PG removal activity of APE1 is highly selective and APE1 is relatively ineffective when the 3′-PG is in singlestranded DNA, at 3′-overhangs or at blunt or recessed 3′-ends of DSBs (27,(29)(30)(31)(32).…”

Human Apurinic/Apyrimidinic Endonuclease 1

“…However, given the many studies which have reported full-length APE1 in the mitochondrial fraction [see for instance (203,213)] and the evidence that the N-terminal portion of APE1 is susceptible to degradation (182,184), it seems likely that non-specific proteolysis occurred during the fractionation steps, leading to the creation of the 33 amino-acid N-terminal truncated form. Since there is no consensus mitochondrial targeting sequence (MTS) in APE1 based on bioinformatics scrutiny, Li et al (118) screened for interactions between a series of peptides that span the length of the APE1 protein and three translocases of the outer mitochondrial membrane. These binding assays uncovered a putative MTS in the C-terminus of APE1, encompassing residues 289-318.…”

Human Apurinic/Apyrimidinic Endonuclease is a Novel Drug Target in Cancer

“…APE1 also appears to have a role in the nucleotide incision repair pathway, where an endonuclease recognises and processes oxidatively-damaged DNA in a glycosylaseindependent manner (Gros et al 2004;Ishchenko et al 2006). A mitochondrial targeting signal in the C-terminal has been identified (Li et al 2010), supporting evidence that APE1 has a role in the repair of oxidatively-damaged mitochondrial DNA (Frossi et al 2002;Shokolenko et al 2009). …”