The aim of the studies is to determine the chemical mechanism by which the mutations in SOD1 lead to FALS and then to determine which of the resultant cellular abnormalities in FALS are shared by sporadic ALS. The applicants' published data show that FALS mutants of SOD1 alter the oxidation-reduction specificity of the SOD1 leading to an enhancement of catalysis of oxidation reactions with peroxides and possibly other oxidants (ONOO-). The proposed work is to determine 1) the limitations of potential electron acceptors and donors for reactions catalyzed by SOD1; 2) the underlying mechanism by which the mutations bring about the altered redox catalysis and 3) alterations in metal binding by the mutant proteins.
Aim 1 (yrs 1-3) proposes to identify the profile of the oxidants where 5 FALS mutants have higher oxidation-reduction reaction rates than WT SOD1 testing 1) t butyl hydroperoxide, 2) other alkyl (lipid) peroxides, 3) peroxynitrite and 4) dioxygen and substrates with 3 substrates for the inactivation reaction (DMPO spin trapping and oxidation of ferrocytochrome or ABTS).
Aim 2 (yrs 1-3) asks what the potential targets of the redox reactions of the FALS mutants can be by structurally and functionally evaluating 1) glutamate transporters (Volterra lab, oxidative modification), 2) neurofilament proteins (Cleveland lab) and structurally only with 3) membrane lipids (Bredesen lab, lipid peroxides).
Aim 3 (year 1) asks, """""""" Can the altered redox chemistry of FALS mutants be attributed to an underlying abnormality in metal binding or migration?"""""""" It seeks to determine a) binding constants for Cu and Zn by equilibrium dialysis and, b) metal migration during redox cycling by spectroscopy.
