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.
"Recent studies have suggested the hypothesis of the involvement of a redox-mediating folding process of APE1 that would affect its subcellular distribution (Li et al. 2010; Vascotto et al. 2011). Increased cellular redox status would result in differential interaction of APE1 with mitochondrial transport proteins increasing the mitochondrial import into mitochondria after increased oxidative stress (Li et al. 2010; Vascotto et al. 2011). "
[Show abstract][Hide abstract] ABSTRACT: Changes in the endocrine system have been suggested to act as signaling factors in the regulation of age-related events. Among the different hormones that have been linked to the aging process, estrogens have been widely investigated. They have been associated with inflammatory and oxidative processes and several investigations have established a relationship between the protective effects of estrogens and the mitochondrial function. Mitochondrial DNA is subjected to continuous oxidative attack by free radicals, and the base excision repair (BER) pathway is the main DNA repair route present in mitochondria. We have investigated the effect of estrogen levels on some of the key enzymes of BER in brain and liver mitochondria. In both tissues, depletion of estrogens led to an increased mitochondrial AP endonuclease (mtAPE1) activity, while restoration of estrogen levels by exogenous supplementation resulted in restitution of control APE1 activity only in liver. Moreover, in hepatic mitochondria, changes in estrogen levels affected the processing of oxidative lesions but not deaminations. Our results suggest that changes in mtAPE1 activity are related to specific translocation of the enzyme from the cytosol into the mitochondria probably due to oxidative stress changes as a consequence of changes in estrogen levels.
"They showed that accumulation of APE1 within mitochondria, as a consequence of oxidative stress, may act as a protective mechanism facilitating mitochondrial genome maintenance and preventing apoptosis  . APE1 is predominantly localized in the nucleus of most cell types, and mitochondrial APE1 is basically scarce and its mitochondrial translocation is largely conditional . On the other hand, Singh et al.  found that mtDNA depletion promotes nuclear genomic instability and that the APE1 expression level is controlled by mtDNA copy number. "
[Show abstract][Hide abstract] ABSTRACT: Maintenance of mitochondrial functionality largely depends on nuclear transcription because most mitochondrial proteins are encoded by the nuclear genome and transported to the mitochondria. Nuclear respiration factor 1 (NRF1) plays a crucial role in regulating the expression of a broad range of mitochondrial genes in the nucleus in response to cellular oxidative stress. However, little is known about the redox regulatory mechanism of the transcriptional activity of NRF1. In this study, we show that the human apurinic/apyrimidinic endonuclease/redox factor (APE1/Ref-1) is involved in mitochondrial function regulation by modulating the DNA-binding activity of NRF1. Our results show that both APE1 expression level and its redox activity are essential for maintenance of the mitochondrial function after tert-butylhydroperoxide-induced oxidative stress. Upon knocking down or redox mutation of APE1, NRF1 DNA-binding activity was impaired and, consequently, the expression of its downstream genes, including Tfam, Cox6c, and Tomm22, was significantly reduced. NRF1 knockdown blocked the restoration of mitochondrial function by APE1 overexpression, which further suggests APE1 regulates mitochondrial function through an NRF1-dependent pathway. Taken together, our results reveal APE1 as a new coactivator of NRF1, which highlights an additional regulatory role of APE1 in maintenance of mitochondrial functionality.
Free Radical Biology and Medicine 05/2012; 53(2):237-48. DOI:10.1016/j.freeradbiomed.2012.04.002 · 5.74 Impact Factor
"Several mtDNA repair proteins, such as TDP1 and PARP-1, appear to lack an MTS (32,40). An MTS has been identified at the C-terminus of APE1 (47), and it appears that the redirection of APE1 into the mitochondria occurs after proteolytic removal by a mitochondria-associated N-terminal peptidase of the first 33 amino acids of APE1 that contains an NLS (29). A putative NLS has been previously identified in PNKP (amino acids 301–304), but it lies in the phosphatase domain of the protein (39,42,48,49) and is presumably retained in the mitochondrial PNKP. "
[Show abstract][Hide abstract] ABSTRACT: Mutations in mitochondrial DNA (mtDNA) are implicated in a broad range of human diseases and in aging. Compared to nuclear DNA, mtDNA is more highly exposed to oxidative damage due to its proximity to the respiratory chain and the lack of protection afforded by chromatin-associated proteins. While repair of oxidative damage to the bases in mtDNA through the base excision repair pathway has been well studied, the repair of oxidatively induced strand breaks in mtDNA has been less thoroughly examined. Polynucleotide kinase/phosphatase (PNKP) processes strand-break termini to render them chemically compatible for the subsequent action of DNA polymerases and ligases. Here, we demonstrate that functionally active full-length PNKP is present in mitochondria as well as nuclei. Downregulation of PNKP results in an accumulation of strand breaks in mtDNA of hydrogen peroxide-treated cells. Full restoration of repair of the H(2)O(2)-induced strand breaks in mitochondria requires both the kinase and phosphatase activities of PNKP. We also demonstrate that PNKP contains a mitochondrial-targeting signal close to the C-terminus of the protein. We further show that PNKP associates with the mitochondrial protein mitofilin. Interaction with mitofilin may serve to translocate PNKP into mitochondria.
Nucleic Acids Research 12/2011; 40(8):3484-95. DOI:10.1093/nar/gkr1245 · 9.11 Impact Factor
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