In this program, we 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 capacity to bind polysomes. Our recent study revealed 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. We found that guanosine in RNA was the predominant ribonucleoside oxidized in cytochrome c (cyt c)-mediated oxidation, while Fe(II)/ascorbate system oxidized all ribonucleoside with no obvious preference. GC/MS and LC/MS analyses demonstrated that the guanine base was not only oxidized but it also depurinated to form an abasic sugar moiety. The aldehyde moieties on the abasic site formed Schiff base with the amino groups in the proteins and generated cross-linking products, such as that between oxidized RNA and cyt c. Interestingly, the formation of the cross-linking product between oxidized RNA and cyt c facilitates the release of cyt c from cardiolipin-containing liposomes, which may represent the release of cyt c from the mitochondria to the cytosol. Thus, the oxidative modification of RNA, including cross-linking, leads not only to impair RNA normal functions, but also to gain a protective signal to facilitate cellular 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, C52, C83, and C173, and the deglutathionylation is catalyzed by sulfiredoxin. 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. In addition, peroxiredoxin I also functions as a molecular chaperone when it exists as a decamer and/or higher molecular weight complexes. We showed that glutathionylation regulates the quaternary structure of peroxiredoxins. Glutathionylation of peroxiredoxin I at C83 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, among them, phosphatase and tensin homolog (PTEN) and mammalian Ste20-like kinase-1 (MST1), 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 and a magnesium and a potasium as cofactors. 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) was found in an opened conformation and was mostly ordered in both structures, with a magnesium bound at the active site of each monomer involving four conserved aspartate residues, D51, D68, D91 and D227. Mutation of these conserved aspartate residues, along with the conserved N87, at the active site, to alanine completely abolished AMP production, highlighting their essential role in the catalytic action of the enzyme. Based on the structural and biochemical analysis of EcSPS reported here, together with the data reported from studies obtained with SPS orthologs from Aquifex aeolicus and humans, a catalytic mechanism was proposed for the selenophosphate synthesis catalyzed by EcSPS.
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