Biological Roles In Mitochondrial DNA Repair And Mitochondria-targeting Mechanism Of Human DNA Repair Enzyme APE1

The elevated level of reactive oxygen species (ROS) in vascular endothelial cells (ECs) has been assumed to participate in initiation and progression of various disorders, particularly that of arthrosclerosis and acute lung injury. Cellular DNA is a biologically important target for ROS. Oxidative DNA damage evoked by ROS, which is mainly produced during oxidative phosphorylation, plays a major role in mitochondrial genomic instability. Mitochondrial DNA (mtDNA), being located in mitochondrial matrix in close proximity to electron transport chain (ETC) and constantly exposed to a high extent of ROS, is extremely susceptible to oxidative damage. In addition, mtDNA is not bound by histones and other associated proteins which can reduce oxidative damage of nuclear DNA (nDNA). It has been discovered that this damage is 10- to 15-fold greater in human mtDNA than that in nDNA in different types of cells. Therefore, mtDNA, as a sentinel molecule in which persistent or severe damage promotes oxidant-mediated cell death, may be a more crucial target for damage by ROS other than nDNA.DNA repair is fundamental to maintenance of genomic integrity. On account of the absence of nucleotide excision repair (NER) in mitochondria, BER, mainly responsible for oxidative and alkylated modifications of individual bases, is supposed to be the major DNA repair pathway in mitochondria. APE1, with dual functions of DNA repair and redox regulation activity of transcription factors, is a rate-limiting enzyme of BER and accounts for nearly all of the AP site incision activity in cell extracts. APE1 exerts repair activity in both nuclei and mitochondria, and its mitochondrial counterpart had been characterized recently as an N-terminal deletion form of full-length APE1. APE1 is the only AP endonuclease in mitochondrion, and loss of mitochondrial APE1, not of the nuclear APE1, is believed to be responsible for triggering apoptosis. It is suggested that mitochondrial APE1 may be a promising therapeutic candidate. Because of a crucial role in cell injury after oxidative stress and limited DNA repair activity of mtDNA, over-expressing DNA repair enzymes in mitochondria represents a potential strategy for preventing even reversing the initiation and progression of oxidative-related diseases. Many recent studies showed that targeting DNA glycosylase in mitochondria resulted in increased cell survival after oxidative stress. However, previous over-expression experiments with heterologous AP endonuclease in mitochondria provided confusing results. Herein we targeted a truncated human APE1 protein specifically in mitochondria of human umbilical vein endothelial cells (HUVECs) by adding mitochondrial targeting sequence (MTS) of human manganese superoxide dismutase (MnSOD). Then we investigated its effect on mtDNA repair capacity and cell survival after oxidative stress.Objective1. To investigate the subcellular distribution alteration of APE1 after oxidative stress induced by IR and hydrogen peroxide.2. To construct plasmid vector expression mitochondrial targeted truncated form of APE1 (pcDNA-mtAPE1-HA) and to investigate the endothelial cell survival and proliferation after peroxide-induced oxidative stress influenced by truncated APE1 mitochodrially overexpression.3. To investigate the presence of MTS in APE1 and its location in the protein sequence of APE1.Materials and Methods1. Alteration of APE1 cellular distribution after oxidative stress: Subcellular distribution of APE1 was examined by laser confocal microscopy at 0-3h in IR treated HOS cell or peroxide treated HUVEC. Western blot was also used to analysis the APE1 protein level in mitochondrial extracts to confirm the morphological observation.2. Construction and biological effects of plasmid vector overexpressing truncated APE1 specifically in mitochondria: First,the N terminal truncated APE1 and MTS was amplified by PCR and spliced by overlap extension PCR then inserted into pcDNA3.1(+) between EcoRI and XhoI sites. Then, the plasmid vector was sequenced and named as pcDNA-mtAPE1-HA. Laser confocal microscopy was used to observe the subcellular localization of recombinant APE1. Western blot and APE assay were applied to measure the increase of APE1 protein and enzymic activity level in mitochondria and nucleus. Subsequently, cellular survival and proliferation was assayed by MTT and colony formation assay separately. Cellular apoptosis was analyzed by FCM and mitochondrial pathway specific apoptosis was investigated by releasing of cytochrome C using western blot.3. Study of presence and localization of MTS in protein sequence of APE1: Constructed eukaryotic expression vector caring truncated form of APE1 in different length. Discriminated the exogenous expressed protein from endogenous APE1 by using different protein tag such as EGFP and HA tag. Subcellular distribution of exogenous expressed truncated APE1 protein was observed by laser confocal microscopy.Results1. Alteration of APE1 cellular distribution after oxidative stress: In untreated human osteosarcoma HOS cell and human endothelial HUVE cell, APE1 was detected mainly in nucleus. Early after treated by 12Gy IR, the significant shifts of APE1 from nucleus to cytoplasm was observed in most HOS cells in the first 3h. The slight shifts of APE1 from nucleus to cytoplasm were observed in hydrogen peroxide treated HUVECs. Western blot results showed that the mitochondrial APE1 level of IR treated HOS cell increased up to 33 fold of control while that of peroxide treated HUVEC increased no more than 5 folds in the first 3 hours ( P<0.05).2. Construction and biological effects of plasmid vector overexpressing truncated APE1 specifically in mitochondria: The recombinant plasmid vector carrying truncated human APE1 confused with MTS at N terminal was constructed successfully and confirmed by sequencing. Recombinant APE1 protein expressed by pcDNA-mtAPE1-HA mainly localized in mitochondria while that expressed by pcDNA-flAPE1-HA mainly localized in nucleus. The APE1 protein level and APE activity significantly increased in mitochondria after pcDNA-mtAPE1-HA transfected. The cellular survival and proliferation after peroxide-induced oxidative stress significantly upregulated in the pcDNA-mtAPE1-HA transfected group (P<0.05), but there are no difference between pcDNA-flAPE1-HA and vector only control group. The enhanced cellular survival benefited from inhibition of mitochondrial specific apoptotic pathway. The pcDNA-mtAPE1-HA transfection effectively suppressed release of cytochrome C, a crucial mitochondrial specific pro-apoptotic agent, from mitochondria to cytoplasm. 3. Study of presence and localization of MTS in protein sequence of APE1: The subcellular distribution style of all the EGFP fusion proteins named pEGFP-42-318-APE1, pEGFP-60-318-APE1, pEGFP-249-318-APE1 and pEGFP-288-318-APE1 were non-specifically diffused in the cell. The HA tagged truncated APE1 expressed by vectors of pEGFP-42-318-APE1-HA, pEGFP-60-318-APE1-HA and pEGFP-249-318-APE1-HA had a mitochondrial specific distribution while the protein expressed by pEGFP-288-318-APE1-HA had a diffused distribution in cells.Conclusion1. The shift of APE1 from nucleus to mitochondria might plays a pivotal role in the cellular response to oxidative stress. The rate and extent of this translocation is determined by the cell type and its sensitivity to the oxidative stress.2. Constructed vector expressing MTS confused truncated APE1 protein mainly increased mitochondrial APE1 protein level and APE activity; vector expressing full length APE1 protein mainly increased nuclear APE1 protein and slightly enhanced nuclear APE activity.3. Mitochondrially overexpression of truncated APE1 effectively enhanced cellular survival and proliferation after oxidative stress, while the overexpression of full length APE1 produced no protection from oxidative stress.4. The survival benefit brought by mitochondrially overexpression of truncated APE1 was by suppression of mitochondrial specific apoptotic pathway.5. Mitochondrial targeting sequence of APE1 was localized in the 249-288 residuals of APE1 protein sequence.6. The C terminal EGFP tag interfered the exposure of the MTS of APE1 by misfolding the C terminal of APE1. The HA tag is the ideal tag for exposure the MTS properly.