共查询到20条相似文献,搜索用时 31 毫秒
1.
Mitochondrial localization of APE/Ref-1 in thyroid cells 总被引:5,自引:0,他引:5
Tell G Crivellato E Pines A Paron I Pucillo C Manzini G Bandiera A Kelley MR Di Loreto C Damante G 《Mutation research》2001,485(2):143-152
2.
The ability to sensitize cancer cells to radiation would be highly beneficial for successful cancer treatment. One mode of action for ionizing radiation is the induction of cell death through infliction of extensive oxidative damage to cellular DNA, including mitochondrial DNA (mtDNA). The ability of cells to repair mtDNA and otherwise maintain the integrity of their mitochondria is vital for protection of the cells against oxidative damage. Because efficient repair of oxidative damage in mtDNA may play a crucial role in cancer cell resistance, interference with this repair process could be an effective way to achieve a radiation sensitive phenotype in otherwise resistant cancer cells. Successful repair of DNA is achieved through a precise and highly regulated multistep process. Expression of excessive amounts of one of the repair enzymes may cause an imbalance of the whole repair system and lead to the loss of repair efficiency. To study the effects of changing mtDNA repair capacity on overall cell survival following oxidative stress, we expressed a bacterial repair enzyme, Exonuclease III (ExoIII) containing the mitochondrial targeting signal of manganese superoxide dismutase, in a human malignant breast epithelial cell line, MDA-MB-231. Following transfection, specific exonuclease activity was found in mitochondrial extracts. In order to examine the effects on repair of oxidative damage in mtDNA, cells were exposed to the enzyme xanthine oxidase and its substrate hypoxanthine. mtDNA repair was evaluated using quantitative Southern blot analysis. The results revealed that cells expressing ExoIII in mitochondria are deficient in mtDNA repair when compared with control cells that express ExoIII without MTS. This diminished mtDNA repair capacity rendered MDA-MB-231 cells more sensitive to oxidative damage, which resulted in a decrease in their long-term survival following oxidative stress. 相似文献
3.
Enhanced mitochondrial DNA repair and cellular survival after oxidative stress by targeting the human 8-oxoguanine glycosylase repair enzyme to mitochondria 总被引:10,自引:0,他引:10
Dobson AW Xu Y Kelley MR LeDoux SP Wilson GL 《The Journal of biological chemistry》2000,275(48):37518-37523
Oxidative damage to mitochondrial DNA (mtDNA) has been implicated as a causative factor in many disease processes and in aging. We have recently discovered that different cell types vary in their capacity to repair this damage, and this variability correlates with their ability to withstand oxidative stress. To explore strategies to enhance repair of oxidative lesions in mtDNA, we have constructed a vector containing a mitochondrial transport sequence upstream of the sequence for human 8-oxoguanine DNA glycosylase. This enzyme is the glycosylase/AP lyase that participates in repair of purine lesions, such as 8-oxoguanine. Western blot analysis confirmed that this recombinant protein was targeted to mitochondria. Enzyme activity assays showed that mitochondrial extracts from cells transfected with the construct had increased enzyme activity compared with cells transfected with vector only, whereas nuclear enzyme activity was not changed. Repair assays showed that there was enhanced repair of oxidative lesions in mtDNA. Additional studies revealed that this augmented repair led to enhanced cellular viability as determined by reduction of the tetrazolium compound to formazan, trypan blue dye exclusion, and clonogenic assays. Therefore, targeting of DNA repair enzymes to mitochondria may be a viable approach for the protection of cells against some of the deleterious effects of oxidative stress. 相似文献
4.
Siddiqui A Rivera-Sánchez S Castro Mdel R Acevedo-Torres K Rane A Torres-Ramos CA Nicholls DG Andersen JK Ayala-Torres S 《Free radical biology & medicine》2012,53(7):1478-1488
Oxidative stress and mitochondrial dysfunction have been implicated in the pathology of HD; however, the precise mechanisms by which mutant huntingtin modulates levels of oxidative damage in turn resulting in mitochondrial dysfunction are not known. We hypothesize that mutant huntingtin increases oxidative mtDNA damage leading to mitochondrial dysfunction. We measured nuclear and mitochondrial DNA lesions and mitochondrial bioenergetics in the STHdhQ7 and STHdhQ111 in vitro striatal model of HD. Striatal cells expressing mutant huntingtin show higher basal levels of mitochondrial-generated ROS and mtDNA lesions and a lower spare respiratory capacity. Silencing of APE1, the major mammalian apurinic/apyrimidinic (AP) endonuclease that participates in the base excision repair (BER) pathway, caused further reductions of spare respiratory capacity in the mutant huntingtin-expressing cells. Localization experiments show that APE1 increases in the mitochondria of wild-type Q7 cells but not in the mutant huntingtin Q111 cells after treatment with hydrogen peroxide. Moreover, these results are recapitulated in human HD striata and HD skin fibroblasts that show significant mtDNA damage (increased lesion frequency and mtDNA depletion) and significant decreases in spare respiratory capacity, respectively. These data suggest that mtDNA is a major target of mutant huntingtin-associated oxidative stress and may contribute to subsequent mitochondrial dysfunction and that APE1 (and, by extension, BER) is an important target in the maintenance of mitochondrial function in HD. 相似文献
5.
Frossi B Tell G Spessotto P Colombatti A Vitale G Pucillo C 《Journal of cellular physiology》2002,193(2):180-186
Reactive oxygen species (ROS) are generated as by-products of respiration and are used as signal transducing intermediates in out-in signaling pathways. ROS are also generated during inflammatory responses and it has been shown that hydrogen peroxide may trigger activation of B-lymphocytes, similar to cross-linking of surface immunoglobulins. On the other hand, both exogenous and endogenous generated ROS are a major source of nuclear and mitochondrial DNA (mtDNA) damage. The base excision repair (BER) enzyme APE/Ref-1 normally repairs small nuclear DNA lesion such as oxidized or alkylated bases. It is not clear though whether DNA repair mechanisms able to abolish oxidative damage from nuclear DNA are present into mitochondria too. Here we show by confocal microscopy and Western blot analysis that in the B-lymphocyte Raji cell line a fraction of APE/Ref-1 rapidly re-localizes into mitochondria following H(2)O(2) activation. Targeting of APE/Ref-1 to mitochondria is not associated with cytochrome-c loss or apoptosis induction. These findings indicate that the APE/Ref-1 translocates to mitochondria in response to oxidative stress and thereby it might exert a protective function. 相似文献
6.
Zhi-Min Zhang Xue-Qin Yang Dong Wang Ge Wang Zhen-Zhou Yang Yi Qing Zhi-Xiang Yang Meng-Xia Li De-Bing Xiang 《Cell biochemistry and biophysics》2011,61(3):561-572
Non-metastatic protein-23 homolog-1 (Nm23-H1) is a multifunctional protein with DNase and histidine protein kinase activities.
Human apurinic endonuclease-1 (APE1) is the AP endonuclease DNA base excision repair (BER) enzyme involved in several important
cellular functions. Since the relationship between Nm23-H1 and APE1 proteins is unclear, we evaluated their interaction at
different time points after irradiating human lung cancer A549 cells with X-rays. We found that Nm23-H1 and APE1 overexpression
was induced by irradiation in a dose- and time-dependent manner. Subcellular distribution pattern of both proteins was reversed
after irradiation. After irradiation, APE1 that initially showed nuclear localization was gradually increased in the cytoplasm,
whereas Nm23-H1 that mainly showed cytoplasmic localization was gradually increased in the nuclei of A549 cells. Nm23-H1 and
APE1 interaction was demonstrated by His-pull-down and co-immunoprecipitation assays. The presence of Nm23-H1/APE1 complex
in X-ray-irradiated A549 cells was also detected by DNA affinity precipitation analysis of a DNA fragment containing an AP
site. Although the AP endonuclease activity of Nm23-H1 was too weak to be detected, the AP endonuclease activity of APE1 was
increased with the enhanced Nm23-H1 expression. In conclusion, our data point to a mechanism by which Nm23-H1 protects cells
against oxidative stress through the engagement of DNA BER enzyme APE1. 相似文献
7.
Reactive oxygen species (ROS) have been implicated as one of the agents responsible for many neurodegenerative diseases. A critical target for ROS is DNA. Most oxidative stress-induced DNA damage in the nucleus and mitochondria is removed by the base excision repair pathway. Apn1 is a yeast enzyme in this pathway which possesses a wider substrate specificity and greater enzyme activity than its mammalian counterpart for removing DNA damage, making it a good therapeutic candidate. For this study we targeted Apn1 to mitochondria in a neuronal cell line derived from the substantia nigra by using a mitochondrial targeting signal (MTS) in an effort to hasten the removal of DNA damage and thereby protect these cells. We found that following oxidative stress, mitochondrial DNA (mtDNA) was repaired more efficiently in cells containing Apn1 with the MTS than controls. There was no difference in nuclear repair. However, cells that expressed Apn1 without the MTS showed enhanced repair of both nuclear and mtDNA. Both Apn1-expressing cells were more resistant to cell death following oxidative stress compared with controls. Therefore, these results reveal that the expression of Apn1 in neurons may be of potential therapeutic benefit for treating patients with specific neurodegenerative diseases. 相似文献
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10.
Narasimhan P Sugawara T Liu J Hayashi T Noshita N Chan PH 《Journal of neurochemistry》2005,93(2):351-358
Oxidative stress after ischemia/reperfusion has been shown to induce DNA damage and subsequent DNA repair activity. Apurinic/apyrimidinic endonuclease (APE) is a multifunctional protein in the DNA base excision repair pathway which repairs apurinic/apyrimidinic sites in DNA. We investigated the involvement of oxidative stress and expression of APE in neurons after oxygen-glucose deprivation and after global cerebral ischemia. Our results suggest that overexpression of human copper/zinc-superoxide dismutase reduced oxidative stress with a subsequent decrease in APE expression. Production of oxygen free radicals and inhibition of the base excision repair pathway may play pivotal roles in the cell death pathway after ischemia. 相似文献
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V. Panduri G. Liu S. Surapureddi J. Kondapalli S. Soberanes N.C. de Souza-Pinto V.A. Bohr G.R.S. Budinger P.T. Schumacker S.A. Weitzman D.W. Kamp 《Free radical biology & medicine》2009,47(6):750-759
8-Oxoguanine DNA glycosylase (Ogg1) repairs 8-oxo-7,8-dihydroxyguanine (8-oxoG), one of the most abundant DNA adducts caused by oxidative stress. In the mitochondria, Ogg1 is thought to prevent activation of the intrinsic apoptotic pathway in response to oxidative stress by augmenting DNA repair. However, the predominance of the β-Ogg1 isoform, which lacks 8-oxoG DNA glycosylase activity, suggests that mitochondrial Ogg1 functions in a role independent of DNA repair. We report here that overexpression of mitochondria-targeted human α-hOgg1 (mt-hOgg1) in human lung adenocarcinoma cells with some alveolar epithelial cell characteristics (A549 cells) prevents oxidant-induced mitochondrial dysfunction and apoptosis by preserving mitochondrial aconitase. Importantly, mitochondrial α-hOgg1 mutants lacking 8-oxoG DNA repair activity were as effective as wild-type mt-hOgg1 in preventing oxidant-induced caspase-9 activation, reductions in mitochondrial aconitase, and apoptosis, suggesting that the protective effects of mt-hOgg1 occur independent of DNA repair. Notably, wild-type and mutant mt-hOgg1 coprecipitate with mitochondrial aconitase. Furthermore, overexpression of mitochondrial aconitase abolishes oxidant-induced apoptosis whereas hOgg1 silencing using shRNA reduces mitochondrial aconitase and augments apoptosis. These findings suggest a novel mechanism that mt-hOgg1 acts as a mitochondrial aconitase chaperone protein to prevent oxidant-mediated mitochondrial dysfunction and apoptosis that might be important in the molecular events underlying oxidant-induced toxicity. 相似文献
13.
DNA base excision repair activities and pathway function in mitochondrial and cellular lysates from cells lacking mitochondrial DNA 总被引:5,自引:0,他引:5
Stuart JA Hashiguchi K Wilson DM Copeland WC Souza-Pinto NC Bohr VA 《Nucleic acids research》2004,32(7):2181-2192
Mitochondrial DNA (mtDNA) contains higher steady-state levels of oxidative damage and mutates at rates significantly greater than nuclear DNA. Oxidative lesions in mtDNA are removed by a base excision repair (BER) pathway. All mtDNA repair proteins are nuclear encoded and imported. Most mtDNA repair proteins so far discovered are either identical to nuclear DNA repair proteins or isoforms of nuclear proteins arising from differential splicing. Regulation of mitochondrial BER is therefore not expected to be independent of nuclear BER, though the extent to which mitochondrial BER is regulated with respect to mtDNA amount or damage is largely unknown. Here we have measured DNA BER activities in lysates of mitochondria isolated from human 143B TK– osteosarcoma cells that had been depleted of mtDNA (ρ0) or not (wt). Despite the total absence of mtDNA in the ρ0 cells, a complete mitochondrial BER pathway was present, as demonstrated using an in vitro assay with synthetic oligonucleotides. Measurement of individual BER protein activities in mitochondrial lysates indicated that some BER activities are insensitive to the lack of mtDNA. Uracil and 8-oxoguanine DNA glycosylase activities were relatively insensitive to the absence of mtDNA, only about 25% reduced in ρ0 relative to wt cells. Apurinic/apyrimidinic (AP) endonuclease and polymerase γ activities were more affected, 65 and 45% lower, respectively, in ρ0 mitochondria. Overall BER activity in lysates was also about 65% reduced in ρ0 mitochondria. To identify the limiting deficiencies in BER of ρ0 mitochondria we supplemented the BER assay of mitochondrial lysates with pure uracil DNA glycosylase, AP endonuclease and/or the catalytic subunit of polymerase γ. BER activity was stimulated by addition of uracil DNA glycosylase and polymerase γ. However, no addition or combination of additions stimulated BER activity to wt levels. This suggests that an unknown activity, factor or interaction important in BER is deficient in ρ0 mitochondria. While nuclear BER protein levels and activities were generally not altered in ρ0 cells, AP endonuclease activity was substantially reduced in nuclear and in whole cell extracts. This appeared to be due to reduced endogenous reactive oxygen species (ROS) production in ρ0 cells, and not a general dysfunction of ρ0 cells, as exposure of cells to ROS rapidly stimulated increases in AP endonuclease activities and APE1 protein levels. 相似文献
14.
Yanlin Jiang Chunlu Guo Melissa L. Fishel Zheng-Yu Wang Michael R. Vasko Mark R. Kelley 《DNA Repair》2009,8(11):1273-1282
Oxidative DNA damage has been implicated in a number of central nervous system pathologies. The base excision repair (BER) pathway is one of the most important cellular protection mechanisms that respond to oxidative DNA damage. Human apurinic (apyrimidinic) endonuclease/redox effector factor (APE1/Ref-1 or APE1) is an essential enzyme in the BER pathway and is expressed in both mitotic and post-mitotic cells in humans. In neurons, a reduction of APE1 expression increases chemotherapy-induced cytotoxicity, while overexpression of APE1 protects cells against the cytotoxicity. However, given the multiple functions of APE1, knockdown of total APE1 is not completely informative of whether it is the redox or DNA repair activity, or interactions with other proteins. Therefore, the use of selective small molecules that can block each function independent of the other is of great benefit in ascertaining APE1 function in post-mitotic cells. In this study, we chose differentiated SH-SY5Y cells as our post-mitotic cell line model to investigate whether a drug-induced decrease in APE1 DNA repair or redox activity contributes to the growth and survival of post-mitotic cells under oxidative DNA damaging conditions. Here, we demonstrate that overexpression of WT-APE1 or C65-APE1 (repair competent) results in significant increase in cell viability after exposure to H2O2. However, the 177/226-APE1 (repair deficient) did not show a protective effect. This phenomenon was further confirmed by the use of methoxyamine (MX), which blocks the repair activity of APE1 that results in enhanced cell killing and apoptosis in differentiated SH-SY5Y cells and in neuronal cultures after oxidative DNA damaging treatments. Blocking APE1 redox function by a small molecule inhibitor, BQP did not decrease viability of SH-SY5Y cells or neuronal cultures following oxidative DNA damaging treatments. Our results demonstrate that the DNA repair function of APE1 contributes to the survival of nondividing post-mitotic cells following oxidative DNA damage. 相似文献
15.
Chouteau JM Obiako B Gorodnya OM Pastukh VM Ruchko MV Wright AJ Wilson GL Gillespie MN 《American journal of physiology. Lung cellular and molecular physiology》2011,301(6):L892-L898
In cultured pulmonary artery endothelial cells and other cell types, overexpression of mt-targeted DNA repair enzymes protects against oxidant-induced mitochondrial DNA (mtDNA) damage and cell death. Whether mtDNA integrity governs functional properties of the endothelium in the intact pulmonary circulation is unknown. Accordingly, the present study used isolated, buffer-perfused rat lungs to determine whether fusion proteins targeting 8-oxoguanine DNA glycosylase 1 (Ogg1) or endonuclease III (Endo III) to mitochondria attenuated mtDNA damage and vascular barrier dysfunction evoked by glucose oxidase (GOX)-generated hydrogen peroxide. We found that both Endo III and Ogg1 fusion proteins accumulated in lung cell mitochondria within 30 min of addition to the perfusion medium. Both constructs prevented GOX-induced increases in the vascular filtration coefficient. Although GOX-induced nuclear DNA damage could not be detected, quantitative Southern blot analysis revealed substantial GOX-induced oxidative mtDNA damage that was prevented by pretreatment with both fusion proteins. The Ogg1 construct also reversed preexisting GOX-induced vascular barrier dysfunction and oxidative mtDNA damage. Collectively, these findings support the ideas that mtDNA is a sentinel molecule governing lung vascular barrier responses to oxidant stress in the intact lung and that the mtDNA repair pathway could be a target for pharmacological intervention in oxidant lung injury. 相似文献
16.
Grishko VI Rachek LI Spitz DR Wilson GL LeDoux SP 《The Journal of biological chemistry》2005,280(10):8901-8905
Numerous studies have revealed that a part of the cellular response to chronic oxidative stress involves increased antioxidant capacity. However, another defense mechanism that has received less attention is DNA repair. Because of the important homeostatic role of mitochondria and the exquisite sensitivity of mitochondrial DNA (mtDNA) to oxidative damage, we hypothesized that mtDNA repair plays an important role in the protection against oxidative stress. To test this hypothesis mtDNA damage and repair was evaluated in normal HA1 Chinese hamster fibroblasts and oxidative stress-resistant variants isolated following chronic exposure to H2O2 or 95% O2. Reactive oxygen species were generated enzymatically using xanthine oxidase and hypoxanthine. When treated with xanthine oxidase reduced levels of initial mtDNA damage and enhanced mtDNA repair were observed in the cells from the oxidative stress-resistant variants, relative to the parental cell line. This enhanced mtDNA repair correlated with an increase in mitochondrial apurinic/apyrimidinic endonuclease activity in both H2O2- and O2-resistant HA1 variants. This is the first report showing enhanced mtDNA repair in the cellular response to chronic oxidative stress. These results provide further evidence for the crucial role that mtDNA repair pathways play in protecting cells against the deleterious effects of reactive oxygen species. 相似文献
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Soumendra Krishna Karmahapatra Tapas Saha Sanjay Adhikari Jordan Woodrick Rabindra Roy 《Molecular and cellular biochemistry》2014,388(1-2):185-193
The Long-Evans Cinnamon (LEC) rat is an animal model for Wilson’s disease. This animal is genetically predisposed to copper accumulation in the liver, increased oxidative stress, accumulation of DNA damage, and the spontaneous development of hepatocellular carcinoma. Thus, this animal model is useful for studying the relationship of endogenous DNA damage to spontaneous carcinogenesis. In this study, we have investigated the apurinic/apyrimidinic endonuclease 1 (APE1)-mediated excision repair of endogenous DNA damage, apurinic/apyrimidinic (AP)-sites, which is highly mutagenic and implicated in human cancer. We found that the activity was reduced in the liver extracts from the acute hepatitis period of LEC rats as compared with extracts from the age-matched Long-Evans Agouti rats. The acute hepatitis period had also a heightened oxidative stress condition as assessed by an increase in oxidized glutathione level and loss of enzyme activity of glyceraldehyde 3-phosphate dehydrogenase, a key redox-sensitive protein in cells. Interestingly, the activity reduction was not due to changes in protein expression but apparently by reversible protein oxidation as the addition of reducing agents to extracts of the liver from acute hepatitis period reactivated APE1 activity and thus, confirmed the oxidation-mediated loss of APE1 activity under increased oxidative stress. These findings show for the first time in an animal model that the repair mechanism of AP-sites is impaired by increased oxidative stress in acute hepatitis via redox regulation which contributed to the increased accumulation of mutagenic AP-sites in liver DNA. 相似文献
19.
Beatriz Ferrando Ana L.D.M. Furlanetto Ricardo Gredilla Jesper F. Havelund Kim H. Hebelstrup Ian M. Mller Tinna Stevnsner 《Physiologia plantarum》2019,166(2):494-512
Mitochondria are one of the major sites of reactive oxygen species (ROS) production in the plant cell. ROS can damage DNA, and this damage is in many organisms mainly repaired by the base excision repair (BER) pathway. We know very little about DNA repair in plants especially in the mitochondria. Combining proteomics, bioinformatics, western blot and enzyme assays, we here demonstrate that the complete BER pathway is found in mitochondria isolated from potato (Solanum tuberosum) tubers. The enzyme activities of three DNA glycosylases and an apurinic/apyrimidinic (AP) endonuclease (APE) were characterized with respect to Mg2+ dependence and, in the case of the APE, temperature sensitivity. Evidence for the presence of the DNA polymerase and the DNA ligase, which complete the repair pathway by replacing the excised base and closing the gap, was also obtained. We tested the effect of oxidative stress on the mitochondrial BER pathway by incubating potato tubers under hypoxia. Protein carbonylation increased significantly in hypoxic tuber mitochondria indicative of increased oxidative stress. The activity of two BER enzymes increased significantly in response to this oxidative stress consistent with the role of the BER pathway in the repair of oxidative damage to mitochondrial DNA. 相似文献
20.
Intracellular trafficking and regulation of mammalian AP-endonuclease 1 (APE1), an essential DNA repair protein 总被引:1,自引:0,他引:1
AP endonuclease (APE), with dual activities as an endonuclease and a 3' exonuclease, is a central player in repair of oxidized and alkylated bases in the genome via the base excision repair (BER) pathway. APE acts as an endonuclease in repairing AP sites generated spontaneously or after base excision during BER. It also removes the 3' blocking groups in DNA generated directly by ROS or after AP lyase reaction. In contrast to E. coli and lower eukaryotes which express two distinct APEs of Xth and Nfo types, mammalian genomes encode only one APE, APE1, which is of the Xth type. However, while the APEs together are dispensable in the bacteria and simple eukaryotes, APE1 is essential for mammalian cells. We have shown that apoptosis of mouse embryo fibroblasts triggered by APE1 inactivation can be prevented by ectopic expression of repair competent but not repair-defective APE1. The mitochondrial APE (mtAPE) is an N-terminal truncation product of APE1. A significant fraction of APE1 is cytosolic, and oxidative stress induces its nuclear and mitochondrial translocation. Such age-dependent increase in APE activity in the nucleus and mitochondria is consistent with the hypothesis that aging is associated with chronic oxidative stress. 相似文献