title: In vivo peroxidative activity of FALS-mutant human CuZnSODs expressed in yeast.
abstract: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder leading to loss of motor neurons. We previously characterized the enhanced peroxidative activity of the human familial ALS (FALS) mutants of copper-zinc superoxide dismutase (CuZnSOD) A4V and G93A in vitro. Here, a similar activity is demonstrated for human FALS CuZnSOD mutants in an in vivo model system, the yeast Saccharomyces cerevisiae. Spin trap adducts of alpha-(pyridyl-4-N-oxide)-N-tert-butylnitrone (POBN) have been measured by electron paramagnetic resonance (EPR) in yeast expressing mutant (A4V, L38V, G93A, and G93C) and wild type CuZnSOD upon addition of hydrogen peroxide to the culture. The trapped radical is a hydroxyethyl adduct of POBN, identified by spectral parameters. Mutant CuZnSODs produced greater concentrations of the trapped adduct compared to the wild type enzyme. This observation provides evidence for an oxidative radical mechanism, whereby the mutants of CuZnSOD catalyze the formation of reactive oxygen species that may be related to the development or progression of FALS. This study also presents an in vivo model system to study free radical production in FALS-associated CuZnSOD mutations.
authors.affiliation: Department of Chemistry and Biochemistry, Loyola Marymount University, Los Angeles, CA 90045-8225, USA. jroe@lmu.edu
authors.forename: James A
authors.initials: JA
authors.lastname: Roe
authors.forename: Martina
authors.initials: M
authors.lastname: Wiedau-Pazos
authors.forename: Vanessa N
authors.initials: VN
authors.lastname: Moy
authors.forename: Joy J
authors.initials: JJ
authors.lastname: Goto
authors.forename: Edith Butler
authors.initials: EB
authors.lastname: Gralla
authors.forename: Joan Selverstone
authors.initials: JS
authors.lastname: Valentine
mesh_terms: D000690:Amyotrophic Lateral Sclerosis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D000431:Ethanol / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D010084:Oxidation-Reduction; D017354:Point Mutation; D011725:Pyridines / Q000032:analysis*; D017382:Reactive Oxygen Species / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D019097:Spin Trapping; D013482:Superoxide Dismutase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
chemical_list: C450079:(pyridyl-4-N-oxide)-N-tert-butylnitrone; D011725:Pyridines; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; C065424:1-hydroxyethyl radical; D000431:Ethanol; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase