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Biological Roles in Mitochondrial DNA Repair and Mitochondria-targeting Mechanism of Human DNA Repair Enzyme APE1

Author: LiMengXia
Tutor: WangDong
School: Third Military Medical University
Course: Pathology
Keywords: Radiotherapy Osteosarcoma Oxidative Stress Vascular endothelial Gene therapy Mitochondria Overexpression DNA repair gene Apurinic / apyrimidinic endonuclease Redox factor-1 Apoptosis Mitochondrial targeting signal
CLC: R363
Type: Master's thesis
Year: 2008
Downloads: 232
Quote: 0
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Abstract


Oxidative stress is closely related with a variety of pathological conditions, including cancer, aging and cardiovascular disease. DNA oxidative damage is one of the important target molecules, formed mainly by oxidative damage to DNA base excision repair (BER) for repair. BER pathway APE1 is rate-limiting enzyme, the level of the expression is essential for the activity of the BER. Mitochondria are the only outside the nucleus in addition to genomic DNA organelles, due to lack of histone protein coding region of dense no intron, combined with the adjacent electron transport chain, mtDNA vulnerable to oxidative stress injury, studies have shown that normal cells mtDNA steady-state level of oxidative damage nDNA of 3-15 times. However, normal cell mtDNA repair activity is weak, through the means of gene therapy to improve mtDNA repair capacity as enhanced cellular resistance to oxidative stress new strategy. Various exogenous increase mitochondrial DNA glycosylase can effectively enhance the expression of mtDNA repair activity in mammalian cells to enhance the survival of oxidative stress. However, due to glycosylase substrate specificity is high, only for a single injury to repair, so more research is focused on the effect on the common pathway of BER repair enzymes APE1, hopes to enhance its activity to achieve a more extensive rehabilitation enhancements effect. Studies have succeeded in mitochondria expressing full-length wild-type gene or APE1 APE1 homologous genes, but no significant effect or produce cells with the expected opposite effect. Recent studies have reported mitochondrial compared with the full-length APE1 lacking the N-terminal sequence was stronger than the full-length APE1 DNA repair activity, but for the lack of typical MTS APE1 this nucleus-encoded genes, their entry into the mitochondria mechanisms are not yet fully understood. Therefore, this study first observed stimulation of oxidative stress in different cell lines of different subcellular distribution of APE1 changes, discussed in oxidative stress in biological effects, and intends to build N-terminal truncated APE1 gene Mitochondria Mitochondrial expression vector to enhance its expression on endothelial cells to explore the ability of mtDNA repair and further observe its effect on cells under oxidative stress effect of apoptosis and proliferation, the last of the mitochondrial targeting signal APE1 study as in mitochondrial gene therapy targeting APE1 provide a theoretical basis and practical strategies. Objective 1 To explore the different processing factors induced oxidative stress conditions different cells APE1 mitochondrial translocation circumstances and differences; 2. Investigate mitochondrial localization truncated APE1 gene expression in human endothelial cell survival after oxidative stress and proliferation effects; 3. investigate mitochondrial targeting signal exists APE1 possible area for further study its mechanism of mitochondrial targeting foundation. Research content and methods. Oxidative stress on APE1 subcellular localization effects: using laser scanning confocal microscopy of FITC-labeled APE1 in oxidative stress changes in intracellular localization, and protein extracted mitochondrial fraction confirmed morphology. Analysis of oxidative stress on APE1 subcellular localization, and with the impact of changes in different cell differences directly for further targeting to mitochondria APE1 provide a theoretical basis for gene therapy. (2) the expression of truncated APE1 mitochondrial targeting vector and its biological effects: the eukaryotic expression plasmid pcDNA3.1 () as the carrier, first obtained by PCR MTS and ND A PE1 sequence, and then SOE obtained through gene splicing, and linearized pcDNA3.1 () plasmid construct connection pcDNA-mtAPE1-HA, by DNA sequencing. Confocal laser scanning microscopy transfected pcDNA-mtAPE1-HA after subcellular localization; Western blot analysis and APE activity detected on HUVE mitochondria and mtDNA repair capacity APE1 level of enhancement; by MTT and clone formation assay oxidative After stimulated cell survival and proliferation, cell apoptosis was detected by flow cytometry and Western blot was used cytosolic cytochrome C release. 3. APE1 mitochondrial localization signal Study: Building C-terminal HA tag with EGFP and different lengths of truncated APE1 fusion protein, the cells were observed with a confocal laser distribution within the presumed mitochondrial targeting signal approximate distribution area. Results 1. Oxidative stress on APE1 subcellular localization effects: within 3 hours after ionizing radiation osteosarcoma cells before irradiation APE1 distributed by the distribution into the nucleus to the cytoplasm based distribution, three hours more than 30-fold increase in mitochondrial APE1 ; H 2 O 2 within 3 hours after treatment of vascular endothelial HUVE cells APE1 only a small amount of translocation into the mitochondria, mitochondrial APE1 within 3 hours of increased levels of no more than 5 times. (2) the expression of truncated APE1 mitochondrial targeting vector and its biological effects: The sequence analysis showed that the recombinant expression vector was successfully constructed pcDNA-mtAPE1-HA and control vector pcDNA-flAPE1-HA. HUVE cells transfected with pcDNA-mtAPE1-HA found significantly increased levels of mitochondrial APE1, and pcDNA-flAPE1-HA levels were significantly increased nuclear APE1, both can slightly increase the level of cytoplasmic APE1. pcDNA-mtAPE1-HA can significantly increase the transfection activity of mitochondrial APE, APE activity without increasing the nucleus; pcDNA-flAPE1-HA which is only slightly increased activity nucleus APE, APE does not increase the activity of mitochondria. At different concentrations H 2 O 2 -induced oxidative stress after, pcDNA-mtAPE1-HA transfected endothelial cells can enhance cell survival and proliferation, while reducing cell wither death rate, while pcDNA-flAPE1-HA and the control group had no significant difference. Further analysis revealed that, pcDNA-mtAPE1-HA transfection inhibits H 2 O 2 treated cytosolic cytochrome C release. 3. APE1 mitochondrial localization signal Study: tagged with EGFP vector pEGFP-(42-318)-APE1, pEGFP-(60-318)-APE1, pEGFP-(249-318)-APE1 and pEGFP-(288 - 318)-APE1 expressed fusion protein in the cells were whole-cell diffuse nonspecific distribution, while the C-terminal HA tag with the expression vector pEGFP-(42-318)-APE1-HA, pEGFP-(60-318) - APE1-HA and pEGFP-(249-318)-APE1-HA-specific mitochondrial localization occurs while pEGFP-(288-318)-APE1-HA was dispersed non-specific whole cell, subcellular distribution form because of this difference occurred amino acid residues in the 249-288 deletion, suggesting that there may be regions of modified mitochondrial targeting signal. Conclusions 1. Early after ionizing radiation treatment, osteosarcoma HOS cell nucleus from a simple expression of APE1 distributed mainly to the cytoplasmic expression changes; while in H 2 O 2 processing early after a small amount of vascular endothelial HUVE cells APE1 translocation from the nucleus to the mitochondria. APE1 mitochondrial oxidative stress in the early post-translocation is a common phenomenon, but its extent and speed of cell lines with different treatment to different factors related difference in sensitivity. 2 by the mitochondrial targeting sequence fused to the N-terminal 33 amino acid sequence of APE1 sequence deletions and eukaryotic expression vector was transfected into endothelial HUVE cells can significantly enhance the level of mitochondrial APE1, while full-length APE1 eukaryotic expression vector was transfected only APE1 can increase the level of the nucleus. 3 truncated APE1 mitochondria-specific overexpression significantly increased vascular endothelial cells in H 2 O 2 -induced oxidative stress survival and proliferation of cell, while length APE1 overexpression on H 2 O 2 -induced cell death without protective effect. 4 truncated APE1 mitochondrial overexpression increases endothelial cell survival after oxidative stress, by blocking the mitochondrial pathway of apoptosis achieved. 5. APE1 mitochondrial localization sequences may be present in the C-terminal 249-288 amino acid residues within the region. 6 constructed with a protein tag fusion protein, C terminal, such as EGFP larger label is likely to affect the exposure of APE1 mitochondrial targeting sequence, and thus interfere with the mitochondrial localization; while small peptide tag such as the HA is able to better exposure of mitochondrial targeting sequence.

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