Elucidation Of Cellular Damage During Exposure To Oxidative Stress

酵素部分的研究旨在阐明在氧化应激下产生细胞损伤的基本机制，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几个方面的研究： (A)研究信使核糖核酸的生理效应。 越来越多的证据表明，活性氧簇(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，以及衰老过程。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。在此之前，我们已经证明，mRNA的适度氧化会导致功能障碍的多肽的产生，包括由于翻译错误而导致的早熟终止短肽。目前，我们正在使用编码牛视紫红质的mRNA作为模型来研究氧化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的mRNA导入HEK293细胞后，导致与抗视紫质抗体发生交叉反应的高分子量低聚物积累，(Ii)氧化的视紫红质mRNA的翻译上调了ER应激转导，如荧光素酶报告实验监测的ATF6的激活，CHOP转录因子、磷酸化的eIF2和ATF4的表达上调，以及caspase-3活性的适度增加。此外，GC/MS分析表明，内质网应激诱导剂thapsigargin处理HEK293细胞后，细胞内Ca(II)浓度一过性增加，细胞内RNA被氧化。因此，thapsigargin可能部分地通过氧化RNA诱导翻译错误导致异常蛋白积累的机制来发挥其对内质网应激的影响。根据这项研究，一种新的由RNA氧化介导的病理生理学相关的内质网应激模型可能在一些与年龄相关的疾病中发挥作用。此外，对氧化RNA的GC/MS分析表明，RNA在体外和体内的氧化都产生了氧化的碱衍生物和基本糖衍生物。后者为RNA氧化提供了比常用的标记8-OH-Guo更准确的标记。 (B)氧化应激在早衰症中的作用及其对衰老的影响。早衰症是一种遗传性疾病，涉及产生层蛋白A的基因突变，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。我们调查了氧化应激是否在疾病的发展中发挥了作用，以及确定层蛋白A的突变是否直接导致了这一过程。我们的初步数据显示，在人的真皮成纤维细胞中，患者成纤维细胞的ATP水平低于对照组。此外，患者样本的细胞内ROS产生水平是对照组的两倍，早熟患者成纤维细胞的蛋白质氧化水平也高于对照组。此外，所用细胞的传代数在观察到的数据中也起着重要作用。利用细胞模型，也进行了用正常Lamin A和孕激素(Progeria中的Lamin A突变体)转基因的小鼠胚胎成纤维细胞的研究。对这些细胞进行了ROS、氧化蛋白和蛋白分解活性的相对水平分析。 (C)多杀性巴氏杆菌毒素诱导的细胞增殖。根据我们早期对caspase-12活性调控的研究结果，我们发现多杀性巴氏杆菌毒素(PMT)是一种细菌蛋白，通过一种未知的机制诱导成纤维细胞的增殖，并下调血清饥饿的野生型胚胎成纤维细胞(MEF)中caspase-12的mRNA和蛋白表达。PMT对细胞增殖的影响和caspase-12的下调是由异源三聚体G蛋白Galpahq/Galpha11介导的。虽然PMT被发现通过mTOR介导的途径诱导S6的磷酸化，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3T3瑞士细胞中，PMT能够以浓度和时间依赖的方式显著降低细胞的ATP水平，而不是静止的未处理细胞。在野生型和Galphaq缺陷的MEF细胞中观察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PMT处理的细胞与未处理的对照细胞相比，S6蛋白的磷酸化水平仍然很高。这些结果表明，PMT激活mTOR通路受S6磷酸化的监控，不受细胞内ATP水平的影响。此外，PMT处理细胞中较低的ATP水平并没有导致AMPK的激活，AMPK是mTOR的负调控因子。PMT对细胞ATP水平的影响促使我们研究PMT对促进性葡萄糖转运蛋白的影响。用识别Glut1和GLUT4的抗体进行的Western印迹分析表明，PMT以时间和浓度依赖的方式刺激Glut1/4基因的表达。此外，PMT对Glut1/4表达的影响不受mTOR途径的抑制剂雷帕霉素的影响。然而，免疫组织化学的初步结果显示，PMT诱导的Glut1蛋白聚集在细胞质中，经血清处理后不会重新定位到血浆膜上。确定观察到的过量基因调控是PMT诱导的低ATP水平的原因还是结果的实验正在进行中。

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.