Bicarbonate Enhances Peroxidation of SOD/ALS Mutants

Summary

Principal Investigator: Balaraman Kalyanaraman
Abstract: Long-term goal: The broad long-term objectives of this revised application are to understand the direct and indirect mechanisms by which human copper, zinc superoxide dismutase (hSOD1) mutants associated with familial and sporadic amyotrophic lateral sclerosis (ALS) disease cause selective toxicity to motor neurons. Hypotheses: (i) Bicarbonate anion (HCO3-) enhances the covalent aggregation of hSOD1 and hSOD1-ALS mutants via carbonate radical anion (CO3 -)- mediated oxidative degradation of tryptophan residue (Trp-32) to kynurenine-type products, and (ii) Transfection of motor neuron cells (NSC-34) and neuroblastoma cells (SH-SY5Y) with familial ALS mutants (e.g., G93A) stimulates ceramide signaling, mitochondrial reactive oxygen species (ROS), transferrin iron uptake, proteasomal dysfunction and apoptosis or programmed death of neuronal cells. Specific Aims: Initially, the direct mechanism of covalent aggregation of isolated hSOD1 and ALS hSOD1 mutant mediated by CO3 - radical will be determined. Next, we will assess mitochondrial generation of ROS, transferrin iron signaling, protein aggregation, proteasome inhibition, and apoptosis in ALS hSOD1-transfected cells. Finally, the influence of ceramide, a bioactive lipid second messenger, on ROS generation in hSOD1WT and hSOD1G93A-transfected cells will be determined. The effects of iron supplementation and mitochondria-targeted spin probes on ALS disease progression and survival in ALS mutant mice will be assessed. Methods: Analytical techniques to be used will include EPR spin-trapping, MALDI-TOF, NMR, and fluorescence. Novel targeted oxidant-specific probes will be used. Significance: The proposed research will merge the oxidation and aggregation hypotheses in ALS SOD1-dependent toxicity using isolated hSOD1 proteins, and explore ceramide-induced oxidant signaling in G93A-transfected cells, and G93A mutant mice. Understanding the molecular basis of ALS SOD1 mutant toxicity will help improve overall strategies for developing effective drug therapy for ALS. Novelty: The use of state-of-the-art analytical techniques coupled with syntheses of mitochondria-targeted spin probes and fluorescent probes should yield new insights on the molecular mechanism for increased toxicity of ALS SOD1 mutants in motor neuron cells.
Funding Period: 2000-07-01 - 2010-01-31
more information: NIH RePORT

Top Publications

  1. pmc HPLC study of oxidation products of hydroethidine in chemical and biological systems: ramifications in superoxide measurements
    Jacek Zielonka
    Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA
    Free Radic Biol Med 46:329-38. 2009
  2. pmc Oxidation of histidine residues in copper-zinc superoxide dismutase by bicarbonate-stimulated peroxidase and thiol oxidase activities: pulse EPR and NMR studies
    Karunakaran Chandran
    Department of Biophysics, Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, United States
    Biochemistry 49:10616-22. 2010
  3. pmc Hydroethidine- and MitoSOX-derived red fluorescence is not a reliable indicator of intracellular superoxide formation: another inconvenient truth
    Jacek Zielonka
    Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA
    Free Radic Biol Med 48:983-1001. 2010
  4. pmc Boronate probes as diagnostic tools for real time monitoring of peroxynitrite and hydroperoxides
    Jacek Zielonka
    Department of Biophysics, Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA
    Chem Res Toxicol 25:1793-9. 2012

Detail Information

Publications4

  1. pmc HPLC study of oxidation products of hydroethidine in chemical and biological systems: ramifications in superoxide measurements
    Jacek Zielonka
    Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA
    Free Radic Biol Med 46:329-38. 2009
    ....
  2. pmc Oxidation of histidine residues in copper-zinc superoxide dismutase by bicarbonate-stimulated peroxidase and thiol oxidase activities: pulse EPR and NMR studies
    Karunakaran Chandran
    Department of Biophysics, Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, United States
    Biochemistry 49:10616-22. 2010
    ..HCO(3)(-)-mediated, selective oxidative modification of histidines in SOD may be relevant to understanding the molecular mechanism of SOD peroxidase and thiol oxidase activities...
  3. pmc Hydroethidine- and MitoSOX-derived red fluorescence is not a reliable indicator of intracellular superoxide formation: another inconvenient truth
    Jacek Zielonka
    Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA
    Free Radic Biol Med 48:983-1001. 2010
    ....
  4. pmc Boronate probes as diagnostic tools for real time monitoring of peroxynitrite and hydroperoxides
    Jacek Zielonka
    Department of Biophysics, Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA
    Chem Res Toxicol 25:1793-9. 2012
    ..This perspective highlights potential applications of boronates and other fluorescent probes to high-throughput analyses of peroxynitrite and hydroperoxides in toxicological studies...