Elucidation Of Cellular Damage During Exposure To Oxidative Stress

阐明暴露于氧化应激期间的细胞损伤

• 批准号: 7594355
• 负责人: EARL R STADTMAN
• 金额: $ 192.59万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7594355
• 关键词: 8-hydroxyguanine; Age; Aging; Area; Caspase; Cell Proliferation; Cells; Complex; Condition; DNA; Disease; Down-Regulation; Drosophila genus; Embryo; Etiology; Exposure to; Fibroblasts; Fluorescence; G Cells; G-substrate; Gene Mutation; Glucose; Growth Factor Receptors; Half-Life; Health; Hela Cells; Hereditary Disease; Heterotrimeric GTP-Binding Proteins; Hydrogen Peroxide; Individual; Interferon Gamma Receptor Beta Chain; Iron; Label; Lamin Type A; Level of Evidence; Ligands; Luciferases; Mammalian Cell; Mediating; Membrane; Messenger RNA; Modeling; Modification; Mus; Mutation; Nuclear; Nucleic Acids; Numbers; Organism; Oxidative Stress; Oxygen; PDGFRB gene; Pasteurella multocida toxin; Pathway interactions; Patients; Phosphorylation; Physiologic pulse; Physiological; Play; Population; Post-Translational Protein Processing; Production; Progeria; Protein Biosynthesis; Protein Kinase; Proteins; Pulse taking; Radiolabeled; Reactive Oxygen Species; Regulation; Reporter; Research; Role; Serine; Serum; Sirolimus; Structural Protein; Transcriptional Activation; Translations; Up-Regulation; Western Blotting; Wild Type Mouse; Yeasts; autocrine; base; caspase 12; cell growth; inhibitor/antagonist; oxidation; paracrine; planetary Atmosphere; polypeptide; radiotracer; research study; response; vector

Our research is directed toward elucidation of basic mechanisms involved in the production of cellular damage during exposure to oxidative stress and the contributions of such damage to aging and disease. Our current research involves studies in the following areas of research: (a) OXIDATIVE MODIFICATION OF mRNA. Because RNAs are less protected by proteins against reactive oxygen species (ROS) than nuclear DNA, they are more susceptible to oxidation. Results of previous studies demonstrated that oxidation of purified luciferase mRNA led to formation of short dysfunctional polypeptides and premature translation capacities. In continuing studies, we have explored the oxidation of polyA mRNA in HEK293 cells under more physiological conditions (5% oxygen atmosphere in 5.5 mM glucose medium and subconfluent cell population). It was found that oxidized nucleic acid bases, including 8-hydroxyguanine (8-OH-Gua), were increased after mRNA synthesis was inhibited by 5,6-Dichloro-1-Beta-D-ribofuranosylbenzimidazole for 6 hr, the average half-life of mRNA. When cells were either transfected with radiolabeled mRNA or pulse-labeled mRNA, there was an increase in the level of labeled 8-OH-Gua, which was dependent on the atmospheric oxygen concentration or iron availability. Moreover, upon incubation, there was a decrease in the translational efficiency of reporter mRNA transfected into HEK293 cells. Thus, the intracellular mRNA is constantly oxidized under physiological culture conditions. As a result, the fidelity of protein synthesis is deteriorated due to mRNA oxidation. (b) ROLE OF OXIDATIVE STRESS IN PROGERIA AND ITS IMPLICATION IN AGING. progeria is a genetic disease that involves mutation of the gene that produces lamin A, a structural protein that, after its synthesis and post-translational modification, is located in the membrane of the fibroblasts from these patients. In previous studies, we demonstrated that the level of ATP in fibroblasts from Progeria patients is lower than in normal fibroblasts and that the level of ROS is higher than in normal individuals. In continuing studies, evidence that the level of ROS is higher in Progeria patients was confirmed by the demonstration that fibroblasts from Progeria patients contain higher levels of protein carbonyl derivatives (RC=O) than are present in normal patients of the same age. As a model to examine the effect of Lamin A mutation (progerin) on cellular activity, HeLa cells were transfected with normal Lamin A or progerin tagged with green fluorescence protein and were induced by treatment with doxycyclin. The HeLa cells were then treated with hydrogen peroxide to examine their response to oxidative stress. The results of experiment show that higher levels of oxidized proteins occur in both the presence and absence of doxycyclin treatment regardless of the type of protein expressed by the cells. This suggests the possibility that overproduction of lamin A and progerin is detrimental to overall cell health. Because hydrogen peroxide led to increased oxidation in both kinds of cells, further experiments are being conducted with normal HeLa cells and HeLa cells with empty vectors. (c) PASTEURELLA MULTOCIDA TOXIN-INDUCED CELL PROLIFERATION. Led by results of our earlier studies on the regulation of caspase activities, we have carried out studies to elucidate the role of Pasteurella multocida toxin (PMT) in the down-regulation of caspase-12 mRNA and proteins in serum-starved, wild-type mouse embryonic fibroblast cells (MEF). We now show that the proliferative effect of PMT and its effect on caspase 12 are both mediated by heterotrimeric G protein Galphaq/Galpha11. To understand the mechanism by which PMT-induced cell proliferation, we investigated the effect of PMT treatment on the mammalian target of rapamycin-sensitive protein kinase (mTOR). We further showed that the proliferative effect of PMT and its effect on caspase12 are both mediated by heterotrimeric G protein G q/G 11. To understand the mechanism by which PMT-induced cell proliferation, we investigated the effect of PMT treatment on the mammalian target of rapamycin (mTOR), a protein kinase that plays a key role in cell growth and proliferation, in both wild MEF and G q/G 11 deficient cells. mTOR is present in diverse organisms, including yeast, Drosophila, and mammalian cells. It consists of two distinctive complexes with very different physiological functions, TORC1 and TORC2. The former is rapamycin-sensitive, while the later is rapamycin-insensitive. Our results showed that PMT is able to stimulate mTOR in wild type MEF as judged by a hefty increase in the phosphorylation of its downstream target molecule, 40S ribosomal polypeptide S6. This increase in S6 protein phosphorylation is not due the up-regulation of S6 protein in treated cells since, using Western blot analysis, both treated and control non-treated cells express roughly the same amount of the nonphosphorylated form of S6 protein. This observation was further validated by the demonstration that PMT is able to induce mTOR phosphorylation at serine 2448 in wild type MEF cells. In contrast, in G q/G 11 deficient MEF cells, PMT failed to activate mTOR suggesting that G q/G 11 is somehow required for PMT-induced mTOR activation. To investigate whether PMT-induced cell proliferation is directly mediated through mTOR we used rapamycin, a specific inhibitor of TORC1 that is known to exert suppressive effect on cell proliferation. Surprisingly, rapamycin did not inhibit PMT-induced cell proliferation in wild type MEF cells. Although rapamycin did not block the mitogenic effect of PMT, it did inhibit the phosphorylation of S6 protein by rapamycin-sensitive mTOR. This result suggests that the proliferative effect of PMT is independent of rapamycin-sensitive mTOR, but does not rule out the involvement of rapamycin-insensitive mTOR pathway in PMT-induced cell proliferation. Interestingly, mTOR activation by PMT did not appear to involve a paracrine or an autocrine mechanism since PMT induced a down-regulation of growth factor receptors, such as PDGFR and IFGR2 or their ligands like IFG1.

我们的研究旨在阐明氧化应激过程中细胞损伤产生的基本机制，以及这种损伤对衰老和疾病的影响。我们目前的研究涉及以下研究领域：