Thermodynamics, kinetics and structural dynamics of amyotrophic lateral sclerosis-associated mutant copper-zinc superoxide dismutases

Intracellular copper, zinc superoxide dismutase (CuZnSOD) is a homodimeric protein located primarily in the cytosol, nucleus and mitochondrial intermembrane space of eukaryotic cells. The primary role of this enzyme is to protect cells from the damaging effects of the superoxide radical through redox chemistry carried out at its copper center. Human CuZnSOD (hSOD) itself also appears be toxic when expressed as one of over 110 mutants associated with the familial form of the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS). A prominent theory of disease pathogenesis is that mutations in hSOD increase the propensity of the protein to form aggregates which are toxic to the motor neuron. Protein inclusions containing hSOD aggregates are found in ALS patients as well as in transgenic mice overexpressing ALS-associated mutant hSODs. Other types of protein aggregates such as amyloid, which are thought to be distinct from ALS aggregates, are implicated as the toxic species responsible for several neurodegenerative disorders such as Parkinsons, Huntingtons and Alzheimer's disease. In many cases the amyloidogenic proteins are associated with mutations which destabilize the native protein and lead to partially unfolded forms which have an increased potential for self-association, leading to amyloid formation. As aggregation of hSOD may also result from partially unfolded forms, biophysical studies have been carried out in order to determine the stability, folding, dynamics and aggregation of ALS-associated mutant hSODs, E100G, G85R, G93A and A4V, chosen to be chemically and structurally diverse. The use of these different proteins is important for understanding the relationship between the properties of mutations and disease characteristics.; The unfolding of the free thiol-free form of hSOD, utilized as a pseudo wild-type, was found to be reversible in both thermally and chemically induced denaturation. Guanidinium chloride (GdmCl)-induced unfolding of fully metallated (holo) pseudoWT hSOD is very slow and required that denaturation curves be incubated for several weeks to ensure the system reached equilibrium. Both kinetic and equilibrium studies in GdmCl indicated the presence of a monomeric intermediate, while thermal denaturation monitored by differential scanning calorimetry (DSC) was 2-state. The holo mutants EI OOG, G85R and G93A decrease the stability and increase the unfolding rate of hSOD mainly by affecting the stability of the monomer with little effect on dimer dissociation. (Abstract shortened by UMI.)...