Investigators in the Section on Metabolic Regulation have focused on the following projects: (i) RNA oxidation. Growing evidence indicates that RNA oxidation is correlated with a number of age-related neurodegenerative diseases, including the recent finding showing that mRNA oxidation occurs early in motor neuron deterioration in ALS. We previously showed that oxidized mRNA causes a reduction of translation fidelity despite the fact that the oxidized mRNA exhibits a similar affinity as non-oxidized mRNA for its binding to polysomes. In our recent study, we showed that in vitro RNA oxidation catalyzed by cytochrome c (cyt c)/H2O2 or by the Fe(II)/ascorbate/H2O2 system yielded different covalently modified RNA derivatives. Our results reveal that the products of RNA oxidation vary with the oxidant used. Guanosine residues are preferentially oxidized by cyt c/H2O2 relative to the Fe(II)/ascorbate/H2O2 system. GC/MS and LC/MS analyses demonstrated that the guanine base was not only oxidized but also depurinated to form an abasic sugar moiety. Results from gel electrophoresis and HPLC analyses show that RNA formed a cross-linked complex with cyt c in an H2O2 concentration-dependent manner. Furthermore, when cyt c was associated with liposomes composed of cardiolipin/phosphatidylcholine, and incubated with RNA and H2O2, it formed cross-linked complexes with the oxidized RNA and dissociated from the liposome. Quantitative analysis reveals that the release of the cyt c from the liposome is facilitated by the formation of an RNAcyt c cross-linked complex. Thus, RNA oxidation may facilitate the release of cyt c from the mitochondrial membrane to induce apoptosis in response to oxidative stress. (ii) Protein glutathionylation in the regulation of peroxiredoxins. Reversible protein glutathionylation, a redox-sensitive regulatory mechanism, plays an important role in cellular regulation, cell signaling, and antioxidant defense. This mechanism is involved in regulating the functions of peroxiredoxins, a family of ubiquitously expressed thiol-specific peroxidase enzymes. We reported earlier that peroxiredoxin I can be glutathionylated at three of its cysteine residues, Cys52, 83, and 173 JBC 284, 23364 (2009). Glutathionylation of peroxiredoxins at their catalytically active cysteines not only provide the reducing equivalents to support their peroxidase activity but also protect peroxiredoxins from irreversible hyperoxidation. We revealed that glutathionylation regulates the quaternary structure of peroxiredoxins. Glutathionylation of peroxiredoxin I at Cys-83 converts the decameric peroxiredoxin to its dimers with the loss of its chaperone activity. The findings that dimer/oligomer structure-specific peroxiredoxin I binding proteins, e.g., PTEN and mammalian Ste20-like kinase-1, regulate cell cycle and apoptosis, respectively, suggest a possible link between glutathionylation and those signaling pathways. (iii) Structural insights into the catalytic mechanism of E. coli selenophosphate synthetase. Selenophosphate synthetase (SPS) catalyzes the synthesis of selenophosphate, the selenium donor for the biosynthesis of selenocysteine and 2-selenouridine residues in seleno-tRNA. Selenocysteine is incorporated into proteins during translation to form selenoproteins which regulate a variety of cellular processes. SPS catalyzes the formation of selenophosphate using ATP and selenide as substrates. In this reaction, the gamma phosphate of ATP is transferred to the selenide to form selenophosphate, while ADP is hydrolyzed to form orthophosphate and AMP. Current knowledge of this enzyme system is derived from studies using the E. coli SPS. To gain the structural insights and the catalytic mechanism of this enzyme, the crystal structure of the C17S mutant of SPS from E. coli (EcSPSC17S) was investigated. EcSPSC17S crystallizes as a homodimer. The dimeric structure in solution of this enzyme was confirmed by analytical ultracentrifugation experiments. Its glycine-rich N-terminal region (residues 1- 47) exists in an opened conformation and was mostly ordered in both structures, with a magnesium cofactor bound at the active site of each monomer involving the conserved aspartate residues. Mutating these conserved residues (D51, D68, D91, and D227) along with N87, also found at the active site, to alanine completely abolished the production of AMP, highlighting their essential role in the catalytic action of the enzyme. Based on the structural and biochemical analysis of EcSPS reported here and using information obtained from similar studies obtained with SPS orthologs from Aquifex aeolicus and humans, a catalytic mechanism was proposed for the selenophosphate synthesis catalyzed by EcSPS. (iv) Dual function of PKC in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced Mn-superoxide dismutase (MnSOD) expression. MnSOD is a primary defensive enzyme against oxidative stress in mitochondria. We previously showed that TPA induces transcriptional activation of human MnSOD mRNA in human lung carcinoma cells, A549, is mediated by PKC-dependent activation of cAMP- responsive element binding protein (CREB)-1/ATF-1-like factors (JBC 274, 37455-37460). Here we showed that MnSOD protein expression was elevated in response to TPA or TNF-alpha;, but not to hydrogen peroxide treatment. TPA-induced generation of reactive oxygen species (ROS) was blocked by pretreatment with the PKC inhibitor BIM as well as by the NADPH oxidase inhibitor DPI, and by siRNA -knockdown of NADPH oxidase components e.g. Rac1, p22phox, p67phox, and NOXO1 in A549 cells and impaired TPA-induced MnSOD expression. To identify the PKC isozyme involved, we constructed a sod2 gene response reporter plasmid, pSODLUC-3340-I2E-C, capable of sensing the effect of TNF-alpha;and TPA, to monitor the effects of PKC isozyme-specific inhibitors and siRNA-induced knockdown of specific PKC isozyme. Our data indicate that TPA-induced MnSOD expression was independent of p53 as reported in the literatures, and the observed TPA effect is most likely mediated by PKC-alpha and -epsilon dependent signaling pathways. Furthermore, siRNA-induced knock-down of CREB and Forkhead box class O (FOXO) 3a led to a reduction in TPA-induced MnSOD gene expression. Together, our results revealed that TPA upregulates, in part, two PKC-dependent transcriptional pathways to induce MnSOD expression. One pathway involves PKC-alpha catalyzed phosphorylation of CREB and the other involves a PKC-mediated the PP2A catalyzed dephosphorylation of Akt at Ser473 which in turn leads to FOXO3a Ser253 dephosphorylation and its activation.

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National Heart, Lung, and Blood Institute
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Abeykoon, Amila H; Noinaj, Nicholas; Choi, Bok-Eum et al. (2016) Structural Insights into Substrate Recognition and Catalysis in OmpB by Protein Lysine Methyltransferases from Rickettsia. J Biol Chem :
Abeykoon, Amila; Wang, Guanghui; Chao, Chien-Chung et al. (2014) Multimethylation of Rickettsia OmpB catalyzed by lysine methyltransferases. J Biol Chem 289:7691-701
Mieyal, John J; Chock, P Boon (2012) Posttranslational modification of cysteine in redox signaling and oxidative stress: Focus on s-glutathionylation. Antioxid Redox Signal 16:471-5
Noinaj, Nicholas; Wattanasak, Rut; Lee, Duck-Yeon et al. (2012) Structural insights into the catalytic mechanism of Escherichia coli selenophosphate synthetase. J Bacteriol 194:499-508
Chae, Ho Zoon; Oubrahim, Hammou; Park, Ji Won et al. (2012) Protein glutathionylation in the regulation of peroxiredoxins: a family of thiol-specific peroxidases that function as antioxidants, molecular chaperones, and signal modulators. Antioxid Redox Signal 16:506-23
Duverger, Olivier; Chen, Susie X; Lee, Delia et al. (2011) SUMOylation of DLX3 by SUMO1 promotes its transcriptional activity. J Cell Biochem 112:445-52
Kim, Ha Kun; Chung, Youn Wook; Chock, P Boon et al. (2011) Effect of CCS on the accumulation of FALS SOD1 mutant-containing aggregates and on mitochondrial translocation of SOD1 mutants: implication of a free radical hypothesis. Arch Biochem Biophys 509:177-85
Chung, Youn Wook; Kim, Ha Kun; Kim, Ick Young et al. (2011) Dual function of protein kinase C (PKC) in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced manganese superoxide dismutase (MnSOD) expression: activation of CREB and FOXO3a by PKC-alpha phosphorylation and by PKC-mediated inactivation of Akt, respective J Biol Chem 286:29681-90
Park, Ji Won; Mieyal, John J; Rhee, Sue Goo et al. (2009) Deglutathionylation of 2-Cys peroxiredoxin is specifically catalyzed by sulfiredoxin. J Biol Chem 284:23364-74
Seo, Jae Ho; Lim, Jung Chae; Lee, Duck-Yeon et al. (2009) Novel protective mechanism against irreversible hyperoxidation of peroxiredoxin: Nalpha-terminal acetylation of human peroxiredoxin II. J Biol Chem 284:13455-65

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