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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

基本信息

批准号：
7734934
负责人：
EARL R STADTMAN
金额：
$ 141.02万
依托单位：
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
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/7734934
关键词：
Affect Aging Aging-Related Process Antibodies Area Bacterial Proteins Biological Biological Assay Bos taurus Cattle Cell Count Cell Nucleus Cell Proliferation Cell model Cells Cellular Membrane Cytoplasm Data Dermal Development Disease Down-Regulation Elevation Embryo Enzymes Etiology Exposure to Fibroblasts Gene Expression Gene Expression Regulation Gene Mutation Glucose Transporter Hereditary Disease Heterotrimeric GTP-Binding Proteins Human Immunohistochemistry In Vitro Lamin Type A Lead Light Luciferases Mediating Messenger RNA Modeling Molecular Weight Monitor Mus Mutation Nucleic Acids Numbers Oxidative Stress Pasteurella multocida toxin Pathway interactions Patients Peptides Phosphorylation Physiological Plasma Play Premature aging syndrome Process Production Progeria Proteins Quality Control RNA RNA marker Reactive Oxygen Species Regulation Relative (related person)Reporter Research Rhodopsin Role Sampling Serum Sirolimus Stress Structural Protein Swiss 3T3 Cells Thapsigargin Time Transducers Transfection Translations Western Blotting age related base caspase 12 caspase-3 human FRAP1 protein in vivo mTOR Inhibitor mutant novel oxidation polypeptide research study sugar transcription factor CHOP

项目摘要

Research in the Enzymes Section 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) Investigating the physiological effects of mRNA. Growing lines of evidence indicate that elevated reactive oxygen species (ROS) and the accumulation of biomacromolecules are involved in the etiology and/or progression of a number of diseases, as well as in the aging process. Among the nucleic acids, RNAs are more susceptible to oxidation than DNAs; therefore, we studied the mechanisms of RNA oxidation and their physiological consequences. Previously, we showed that moderate oxidation of mRNA leads to production of dysfunctional polypeptides, including prematuree terminated short peptides due to translation errors. Currently, we are using an mRNA-encoding bovine rhodopsin as a model to investigate the biological impact of oxidized mRNA-induced translation errors on protein quality control. Our results demonstrate that (i) transfection of the in vitro synthesized rhodopsin oxidized mRNA into HEK293 cells caused an accumulation of high molecular weight oligomeric species that cross reacted with anti-rhodopsin antibody and (ii) translation of the oxidized rhodopsin mRNA up-regulated the ER stress transducers, such as ATF6 activation monitored by the luciferase reporter assay, and elevation of CHOP transcription factor, phosphorylated eIF2 and ATF4 expression, as well as a moderate increase in caspase-3 activity. In addition, GC/MS analysis revealed that treatment of HEK293 cells with thapsigargin, an ER stress inducer, caused a transient increase in cytosolic Ca(II) and induced cellular RNA oxidation. Thus, thapsigargin may, in part, exert its effect on ER stress via a mechanism mediated by oxidized RNA-induced accumulation of aberrant proteins due to translation errors. In light of this study, a novel pathophysiologically relevant ER stress model mediated by RNA oxidation could, in part, play a role in a number of age-related diseases. In addition, GC/MS analysis of oxidized RNA revealed that in vitro and in vivo oxidation of RNA yielded both oxidized base derivatives and abasic sugar derivatives. The latter provides a more accurate marker for RNA oxidation than 8-OH-Guo, a commonly used marker. (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 associated with the membrane of the cellular nucleus. Patients suffering from this disease show premature aging and generally die before reaching adulthood. We investigated whether oxidative stress plays a role in the development of the disease as well as determining if the mutation of lamin A is directly responsible for this process. Our preliminary data showed that in human dermal fibroblasts the ATP level was lower in the patients fibroblasts than that in the controls. In addition, the level of intracellular ROS production in patient samples was twice that found in the controls and protein oxidation in Progeria fibroblasts was also higher than the controls. Furthermore, the passage number of the cells used also played an important role in the data observed. Using a cell model, studies were also carried out with mouse embryonic fibroblasts transfected with normal Lamin A and progerin, the Lamin A mutant in Progeria. These cells were analyzed with respect to the relative levels of ROS, oxidized proteins, and the proteolytic activity. (c) Pasteurella multocida toxin-induced cell proliferation. Led by results of our earlier studies on the regulation of caspase-12 activities, we showed that Pasteurella multocida toxin (PMT),a bacterial protein known to induce, via an unknown mechanism, fibroblast cell proliferation also down-regulates caspase-12 mRNA and protein in serum-starved, wild-type embryonic fibroblast cells (MEF). The effects of PMT on cellular proliferation and down-regulation of caspase-12 are mediated by a heterotrimeric G protein, Galpahq/Galpha11. While PMT was found to induce S6 phosphorylation via a mTOR-mediated pathway, its effect on proliferation is independent of both mTOR activity and S6 phosphorylation. We showed for the first time that in quiescent 3T3 Swiss cells, PMT is able to significantly lower cellular ATP levels in a concentration- and time-dependent fashion relative to quiescent non-treated cells. The effect of PMT on cellular ATP was observed in wild-type and Galphaq-deficient MEF cells. It is worth noting that, notwithstanding the negative effect of PMT on cellular ATP, PMT-treated cells still showed high levels of S6 protein phosphorylation as compared to control non-treated cells. These results showed that the activation of the mTOR pathway by PMT, monitored by S6 phosphorylation, is not affected by cellular ATP levels. In addition, the lower levels of ATP in PMT-treated cells did not lead to activation of AMPK, a negative regulator of mTOR. The observed effect of PMT on cellular ATP levels prompted us to study the effect of PMT on facilitative glucose transporters. Western blot analysis using antibodies that recognize Glut1 and Glut4 showed that PMT stimulates Glut1/4 gene expression in a time- and concentration- dependent manner. In addition, the effect of PMT on Glut1/4 expression was not affected by rapamycin, an inhibitor of the mTOR pathway. However, preliminary results using immunohistochemistry showed that Glut1 protein induced by PMT accumulated in the cytoplasm and did not relocate to the plasma mambrane following serum treatment. Experiments to determine whether the observed Glut gene regulation is a cause or a consequence of the low ATP levels induced by PMT are ongoing.

酶部分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贡献。我们目前的研究涉及以下研究领域的研究： (a)研究mRNA的生理作用。越来越多的证据表明，活性氧（ROS）的升高和生物大分子的积累参与了许多疾病的病因和/或进展，以及在衰老过程中。在核酸中，RNA比DNA更容易氧化;因此，我们研究了RNA氧化的机制及其生理后果。以前，我们表明，mRNA的中度氧化导致产生功能失调的多肽，包括prematuree终止短肽由于翻译错误。目前，我们正在使用的mRNA编码的牛视紫红质作为一个模型，研究氧化的mRNA诱导的翻译错误对蛋白质质量控制的生物学影响。我们的结果表明，（i）将体外合成的视紫红质氧化mRNA转染到HEK 293细胞中引起与抗视紫红质抗体交叉反应的高分子量寡聚物物质的积累，和（ii）氧化视紫红质mRNA的翻译上调ER应激转导物，例如由荧光素酶报告基因测定监测的ATF 6活化，和CHOP转录因子的升高，磷酸化eIF 2和ATF 4表达，以及caspase-3活性的中度增加。此外，GC/MS分析显示，用毒胡萝卜素（一种ER应激诱导剂）处理HEK 293细胞，引起胞质Ca（II）的瞬时增加，并诱导细胞RNA氧化。因此，毒胡萝卜素可能，部分地，通过氧化RNA诱导的异常蛋白质的积累，由于翻译错误介导的机制发挥其对ER应激的作用。根据这项研究，一种新的病理生理相关的ER应激模型介导的RNA氧化，可以在一定程度上发挥作用，在一些年龄相关的疾病。此外，氧化的RNA的GC/MS分析显示，在体外和体内的RNA氧化产生氧化的碱衍生物和脱碱基糖衍生物。后者为RNA氧化提供了比常用标记物8-OH-Guo更准确的标记物。 (b)氧化应激在早衰症中的作用及其在衰老中的意义。早衰症是一种遗传性疾病，涉及产生核纤层蛋白A的基因突变，核纤层蛋白A是一种与细胞核膜相关的结构蛋白。患有这种疾病的患者表现出过早衰老，通常在成年前死亡。我们研究了氧化应激是否在疾病的发展中起作用，以及确定核纤层蛋白A的突变是否直接导致这一过程。我们的初步数据表明，在人皮肤成纤维细胞中，患者成纤维细胞中的ATP水平低于对照组。此外，患者样品中细胞内ROS产生的水平是对照中发现的两倍，并且早衰症成纤维细胞中的蛋白质氧化也高于对照。此外，所用细胞的传代次数在观察到的数据中也起重要作用。使用细胞模型，也进行了研究与小鼠胚胎成纤维细胞转染正常核纤层蛋白A和早老蛋白，核纤层蛋白A突变体在早衰症。分析这些细胞的ROS、氧化蛋白和蛋白水解活性的相对水平。 (c)多杀性巴氏杆菌毒素诱导的细胞增殖。我们早期的研究结果的caspase-12活性的调节，我们表明，多杀性巴氏杆菌毒素（PMT），一种已知的细菌蛋白诱导，通过一种未知的机制，成纤维细胞增殖也下调caspase-12的mRNA和蛋白在血清饥饿，野生型胚胎成纤维细胞（MEF）。PMT对细胞增殖和caspase-12下调的影响由异源三聚体G蛋白Galpahq/Galpha 11介导。虽然发现PMT通过mTOR介导的途径诱导S6磷酸化，但其对增殖的影响不依赖于mTOR活性和S6磷酸化。我们首次表明，在静止的3 T3瑞士细胞，PMT是能够显着降低细胞ATP水平的浓度和时间依赖性的方式相对于静止的未处理的细胞。在野生型和Galphaq缺陷型MEF细胞中观察到PMT对细胞ATP的影响。值得注意的是，尽管PMT对细胞ATP有负面影响，但与对照未处理的细胞相比，PMT处理的细胞仍显示出高水平的S6蛋白磷酸化。这些结果表明，通过S6磷酸化监测的PMT对mTOR通路的激活不受细胞ATP水平的影响。此外，PMT处理的细胞中较低水平的ATP并不导致AMPK的激活，AMPK是mTOR的负调节因子。观察到的PMT对细胞ATP水平的影响促使我们研究PMT对易化葡萄糖转运蛋白的影响。使用识别Glut 1和Glut 4的抗体的Western印迹分析表明，PMT以时间和浓度依赖性方式刺激Glut 1/4基因表达。此外，PMT对Glut 1/4表达的影响不受mTOR通路抑制剂雷帕霉素的影响。然而，使用免疫组织化学的初步结果表明，谷氨酸1蛋白诱导PMT积累在细胞质中，并没有搬迁到质膜血清处理后。实验，以确定是否观察到的谷氨酸基因调控是一个原因或PMT诱导的低ATP水平的后果正在进行中。