Elucidation Of Cellular Damage During Exposure To Oxidative Stress

酶科的研究旨在阐明在暴露于氧化应激期间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研究领域：

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.