Copper-zinc superoxide dismutase and amyotrophic lateral sclerosis: Solution dynamics of the apoproteins and shotgun proteomics of SOD1 aggregates purified from transgenic mouse

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that involves the selective death of motor neurons. Nearly 2% of ALS cases are caused by mutations in the gene encoding copper-zinc superoxide dismutase (SOD1). These SOD1 mutations cause ALS by imparting a toxic function to the protein, in addition to its normal, beneficial function as a superoxide anion scavenger. An abundance of experimental evidence points towards SOD1 aggregation as the underlying cause of SOD1 neurotoxicity and it is thought that pathogenic mutations to SOD1 increase the aggregation propensity of the SOD1 polypeptide. This thesis reports hydrogen-deuterium (H/D) exchange and differential scanning calorimetry (DSC) data that demonstrates ALS mutations to SOD1 do not uniformly destabilize the native fold of the polypeptide. Moreover, many ALS mutations do not perturb any studied property of SOD1 such as metal coordination or SOD activity. Hence, destabilization of the SOD1 native state is insufficient to completely explain the neurodegenerative effects of ALS mutations in SOD1. Additionally, this thesis presents site-specific H/D exchange data on the metal free (apo) form of the A4V variant of SOD1. The A4V mutation is the most frequently occurring ALS-linked mutation to SODI. H/D exchange monitored by mass spectrometry revealed that many regions of the folded SOD1 polypeptide were not perturbed by the A4V substitution. However, one region of the protein, residues 50-53, of the disulfide sub-loop and dimer interface, exhibited no protection from H/D exchange in the A4V apoprotein and this was in contrast to the wild-type (WT) SOD1 protein. A subpopulation of A4V apo-SOD1 was also observed to undergo slow, localized unfolding at residues 21-53, which represents beta-strands 3 and 4. Upon reduction of the intramolecular disulfide bond, A4V apo-SOD1 exhibited no detectable endothermic transition with DSC and exchanged with D2O rapidly, at a rate characteristic of a random coil polypeptide. This thesis also describes a sequential size exclusion/immunoaffinity chromatography method for purifying SOD1 and SOD1 aggregates from the spinal cord of transgenic mice carrying the human SOD1 gene with various ALS mutations. A mass spectrometry based proteomic analysis of purified samples revealed that calmodulin-3, glial fibrillary acidic protein, neurofilament, and various membrane proteins from the mitochondrial electron transport chain were present with mutant hSOD1 in high molecular weight (>70 kDa) complexes. Additionally, MALDI-TOF MS showed that various mutant SOD1 proteins derived from these high molecular weight complexes also possessed apparent low molecular weight modifications (<100 Da). In lower molecular weight complexes (<70 kDa), mutant SOD1 was observed to be associating with Bcl-2 associated protein-X (BAX).