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.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Intramural Research (Z01)
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National Heart, Lung, and Blood Institute
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Noda, Yasuko; Berlett, Barbara S; Stadtman, Earl R et al. (2007) Identification of enzymes and regulatory proteins in Escherichia coli that are oxidized under nitrogen, carbon, or phosphate starvation. Proc Natl Acad Sci U S A 104:18456-60
Tanaka, Mikiei; Chock, P Boon; Stadtman, Earl R (2007) Oxidized messenger RNA induces translation errors. Proc Natl Acad Sci U S A 104:66-71
Miyoshi, Noriyuki; Oubrahim, Hammou; Chock, P Boon et al. (2006) Age-dependent cell death and the role of ATP in hydrogen peroxide-induced apoptosis and necrosis. Proc Natl Acad Sci U S A 103:1727-31
Khan, Mohammed A S; Chock, P Boon; Stadtman, Earl R (2005) Knockout of caspase-like gene, YCA1, abrogates apoptosis and elevates oxidized proteins in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 102:17326-31
Stadtman, Earl R; Van Remmen, Holly; Richardson, Arlan et al. (2005) Methionine oxidation and aging. Biochim Biophys Acta 1703:135-40
Lee, Byung Cheon; Lee, Yong Kwon; Lee, Ho-Joung et al. (2005) Cloning and characterization of antioxidant enzyme methionine sulfoxide-S-reductase from Caenorhabditis elegans. Arch Biochem Biophys 434:275-81
Stadtman, Earl R; Arai, Hirofumi; Berlett, Barbara S (2005) Protein oxidation by the cytochrome P450 mixed-function oxidation system. Biochem Biophys Res Commun 338:432-6
Oubrahim, Hammou; Wang, Jun; Stadtman, Earl R et al. (2005) Molecular cloning and characterization of murine caspase-12 gene promoter. Proc Natl Acad Sci U S A 102:2322-7
Arai, Hirofumi; Berlett, Barbara S; Chock, P Boon et al. (2005) Effect of bicarbonate on iron-mediated oxidation of low-density lipoprotein. Proc Natl Acad Sci U S A 102:10472-7
Williams, W M; Stadtman, E R; Moskovitz, J (2004) Ageing and exposure to oxidative stress in vivo differentially affect cellular levels of PrP in mouse cerebral microvessels and brain parenchyma. Neuropathol Appl Neurobiol 30:161-8

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