Free radical and reactive oxygen species (ROS) play important roles in the etiology and/or progression of a number of diseases and in aging as well as in signal transduction. Investigators in the Section on Metabolic Regulation carried out studies to elucidate mechanisms by which free radicals and ROS are generated and exert their biological effects. During this fiscal year, our research was focused on: (i) Protein sumoylation plays an important role in ROS-induced stress. Using proteomic methodology and the construction of controllable HEK 293 Tet-on stable cell lines, we overexpressed Myc-SUMO-1 to amplify the formation of SUMO-1 sumoylated proteins in order to identify them?particularly, those normally existing in relatively low quantities. Overexpression of SUMO-1 does not significantly alter cell growth and the modified proteins mainly occurred in the nucleus, which is consistent with that observed in control cells. The sumoylated proteins were affinity purified or isolated by immunoprecipitation and identified by sequence analysis using mass spectrometric methods. Our results revealed that several heterogeneous nuclear ribonucleoproteins (hnRNP), zinc finger proteins, and nuclear pore complex proteins were sumoylated. Since hnRNPs have been implicated in RNA splicing, transport, stability, and translation, our observation suggests that sumoylation may play an important role in regulating mRNA metabolism. The functional effects of these hnRNPs by sumoylation are currently under investigation. In addition, overexpression of SUMO-2/3 causes a drastic reduction in growth rate that appears to be linked to p53 modification. (ii) Previously, we developed a stable and controllable RNAi method that generated dsRNA intracellularly. This method was extended to animal studies and to knock down catalase, Cu/Zn superoxide dismutase, and Mn-superoxide dismutase in cells to investigate how these anti-oxidant enzymes affect cellular cell signaling, apoptosis, and senescence. To this end, we attempted to study the mechanisms of RNA interference?in particular, finding proteins that are involved in RISC, a P-32- and photoaffinity-moiety-labeled 22 bp dsRNA fragment that was synthesized and used to fish out protein(s) that directly interact with the dsRNA fragment. The protein that binds to the dsRNA was identified as TB-RBP/translin. Further analysis of the DNA/RNA-binding protein revealed that it possesses both ssRNase and dsRNase activities. It processes long dsRNA into mainly ~25 bp fragments by binding to the open ends of dsRNA and hydrolyzing it with limited turnover due to its high affinity towards the products. Both ssRNase and dsRNase activities are inhibited by common RNase inhibitors. We are currently searching for regulatory units that control translin activities in vivo. (iii) Earlier we showed that serum deprivation leads to elevation of ROS levels and apoptosis in neuroblastoma SH-SY5Y cells. Hormesis and anti-apoptotic effects were also observed. These effects are mediated by the cyclic GMP-dependent protein kinase pathway that leads to increased expression of thioredoxin, Mn-superoxide dismutase, and anti-apoptotic Bcl-2. Further studies revealed that the elevated expression of Bcl-2, in part, is mediated by redox-dependent activation of cyclic AMP-dependent protein kinase that phosphorylated CREB. (iv) We previously showed that the Mn(II) complex could function as an anti-oxidant and at high concentrations, Mn(II) induced caspase-12 mediated apoptosis. We have now cloned and characterized the murine caspase-12 gene promoter. To follow up our studies on Cu/Zn superoxide dismutase mutants of familial amyotrophic lateral sclerosis, recent result indicate that Golgi apparatus and/or secretory pathways are likely the early targets of FALS mutant toxicity. In addition, the effect of RNA oxidation on translational efficiency was also investigated. (v) Previously, we built an instrument to study the effects of applying external electric fields on the integrity of the cell membrane and to investigate cellular events. This method was extended using a larger external electric pulse of sub-microsecond duration with a primary aim of achieving selective permeabilization in a hetergenous population of vesicles or cells containing membrane vacuoles.
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