During this fiscal year, investigators in the Section on Metabolic Regulation have focused on the following projects: (i) Protein sumoylation has been shown to play a role in oxidative stress. To elucidate the enzymatic pathways and identify target proteins for this family of ubiquitin-like modifiers, such as SUMO-2, SUMO-3, NEDD8, and FAT10, a general proteomic approach was developed. It involved establishing stable HEK293 Tet-On cell lines for expressing the epitope-tagged ubiquitin-like modifier proteins and their mutants. The epitope-tagged proteins were purified and analyzed using liquid chromatography coupled with mass spectrometry. The results revealed a number of new target proteins for SUMO-2, SUMO-3, NEDD8, and FAT10. Among them, p53 was found to form a covalent complex with SUMO-2, SUMO-3, and FAT10. Cells overexpressing SUMO-2, SUMO-3, or FAT10 led to upregulation of p53 activity. This finding shows that p53 is the first identified FATylated protein. In addition, the conformation of p53 is altered and its transcriptional activity is greatly elevated in FAT10 overexpressing cells. Overexpressing FAT10 also leads to a change in the size and distribution of PML nuclear bodies. In addition, stable cell lines overexpressing the SUMO conjugating enzyme, UBC9, as well as that overexpressing its dominant-negative mutant (UBCO-DN) have also been established. These stable cell lines will be used to study the functional changes in cells with the upregulation or downregulation of SUMOylation.? ? (ii) ROS are known to oxidize cellular macromolecules, such as proteins, lipids, and nucleic acids, which leads to the loss of their physiological functions. Little attention has been focused on RNA oxidation. Relative to genomic DNAs, RNAs, are less protected by proteins; therefore, they are more vulnerable to ROS-mediated oxidative modification. We previously showed that oxidation of purified luciferase mRNA lead to significant reduction of its translation fidelity. In continuing studies, we investigated the oxidation of polyA mRNA in HEK293 cells under more physiological conditions, i.e. 5% oxygen atmosphere in 5.5 mM glucose medium and subconfluent population. We detected that oxidized nucleic acid bases, including 8-OH-Gua, were elevated after mRNA synthesis was inhibited for 6 hr, the average half-life of mRNA, by 5,6-dichloro-1-beta-D-ribofuraosylbenzimidazole. When cells were either transfected with radiolabeled mRNA or pulse-labeled mRNA, the level of labeled 8-OH-Gua increased as a function of oxygen concentration or iron availability. Correspondingly, there was a decrease in translational efficiency of the reporter mRNA transfected into HEK293 cells. These results reveal that intracellular mRNA is constantly oxidized under physiological conditions and causes translational errors.? ? (iii) Manganese superoxide dismutase (MnSOD) is a highly regulated member of SODs, in response to oxidative stress. The free radical-mediatedspecifically, the superoxide radical anion-mediated signal transduction pathway--induction of MnSOD in A549 cells in response to TPA stress was investigated. Our previous study, we found that TPA-responsive induction of the MnSOD gene requires binding of the CRAB/ATF-1-like transcription factor to the MSTRE (MnSOD TPA Responsive Element) in the promoter region of the human MnSOD gene. This induction also requires the activation of PKC and NADPH oxidase, indicating the involvement of superoxide radical generation as an upstream signal. Further study indicated that PKC-alpha-mediated phosphorylation of CRAB-like factor is also required. In addition to the MSTRE binding site, FoxO-3a sites were also identified in the promoter. FoxO transcription factors are emerging as an important family of proteins that modulate the expression of genes involved in apoptosis, the cell cycle, DNA damage repair, oxidative stress, cell differentiation, glucose metabolism, and longevity. We are investigating the contribution of FoxO-3a binding in the promoter to the TPA-induced MnSOD. Preliminary results indicate that FoxO-3a is also overexpressed as well as phosphorylated during MnSOD induction in response to the TPA stress. Moreover, the FoxO-3a induction and phosphorylation is controlled by PKC-deltat. We are continuing to elucidate the superoxide-mediated signal pathways by monitoring MnSOD induction.? ? ? (iv) Missense mutations in the coding regions of the CuZnSOD (SOD1) gene have been linked to familial amyotrophic lateral sclerosis (FALS). It is believed that FALS SOD1 mutants act through gain of cytotoxic function(s), including the generation of SOD1-containing aggregates. Previously, we showed that two FALS mutants enhance catalytic activity for free radical generation, which could facilitate formation of protein aggregates. To investigate the role of the copper chaperon for SOD1 (CCS) in forming SOD1-containing aggregates, we expressed mutant SOD1 and CCS together from dicistronic mRNA in HEK293 cells. Our results show that overexpressed mutant SOD1, but not wild type SOD1, from monocistronic mRNA generates detergent-insoluble SOD1 mutants containing aggregates. In contrast, mutant SOD1 co-expressed with CCS from dicistronic mRNA showed a drastic reduction of the SOD1 mutants containing aggregates. This CCS effect was neutralized extensively when cells were pretreated with a proteasome inhibitor or an inhibitor for the autophagy process.? ? (v) Accumulation of ROS has been linked to Alzheimers disease via the formation of superoxide radical anions mediated by Beta-amyloid peptides of 39 to 42 amino acid residues derived from amyloid precursor protein, APP. We have established a SH-SY5Y cell model to study the mechanism by which the peptide A-beta 42 induces the generation of superoxide radical anion.? ? (vi) Cells undergoing apoptosis lose lipid asymmetry such that phosphatidylserine (PS) is exposed to the outer surface of the cell membrane. Macrophages, as well as many other cells, recognize PS via a PS-receptor to signal phagocytosis of the PS displaying host cells thereby eliciting a non-inflammatory response. PS externalization could also be effected via short-lived membrane pores induced by external electric fields in normal non-apoptotic cells. A Molecular Dynamics simulation study of a mixed DOPC/DOPS lipid bilayer with 10% asymmetric DOPS showed that both reversible membrane pores and PS externalization are possible. Cells induced to display PS by this field method could thus be targeted as apoptotic and be subject to phagositic clearance by the immune system. We tested this method by using a mouse macrophage cell line (J7444A.1) and a mouse B-Cell line (FOX-NY) from the same strain and find that, in the presence of Annexin I, a significant ( 30%) engulfment of the field treated B-cell by macrophages compared to the control non-field treated cells.? ? (vii) The Molecular Dynamics simulation was used to study the molecular details involved in membrane electroporation and resealing, field-induced fusion between two bilayer membranes, and ion transport through membrane pores. We are using DOPC or DOPC/DOPS lipid bilayer mixtures with or without ions (NaCl) in the aqueous medium as model systems. These studies are currently ongoing and we aim to address relevant questions in the field such as: Why and how are pores formed by the field, i.e. the energetics of the process; how are ions transported across the lipid bilayer; what governs the resealing process and does it entail new lipid structures as observed experimentally; and how does field induced membrane fusion occur, and how is it different from other known fusion pathways, such as stalk formation and hemifusion.
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