Structural similarity of wild-type and ALS-mutant superoxide dismutase-1 fibrils using limited proteolysis and atomic force microscopy

Abstract

Abnormal assemblies formed by misfolded superoxide dismutase-1 (SOD1) proteins are the likely cause of SOD1-linked familial amyotrophic lateral sclerosis (fALS) and may be involved in some cases of sporadic ALS. To analyze the structure of the insoluble SOD1 amyloid fibrils, we first used limited proteolysis followed by mass spectrometric analysis. Digestion of amyloid fibrils formed from full-length N-acetylated WT SOD1 with trypsin, chymotrypsin, or Pronase revealed that the first 63 residues of the N terminus were protected from protease digestion by fibril formation. Furthermore, every tested ALS-mutant SOD1 protein (G37R, L38V, G41D, G93A, G93S, and D101N) showed a similar protected fragment after trypsin digestion. Our second approach to structural characterization used atomic force microscopy to image the SOD1 fibrils and revealed that WT and mutants showed similar twisted morphologies. WT fibrils had a consistent average helical pitch distance of 62.1 nm. The ALSmutant SOD1 proteins L38V, G93A, and G93S formed fibrils with helical twist patterns very similar to those of WT, whereas small but significant structural deviations were observed for the mutant proteins G37R, G41D, and D101N. Overall, our studies suggest that human WT SOD1 and ALS-mutants tested have a common intrinsic propensity to fibrillate through the N terminus and that single amino acid substitutions can lead to changes in the helical twist pattern.aggregation | mass spectrometry | protein misfolding | neurodegeneration A myotrophic lateral sclerosis (ALS) or Lou Gehrig's disease is a devastating motor neuron disease characterized by the formation of abnormal protein aggregates in neuronal cells. More than 100 different mutations in superoxide dismutase-1 (sod1) have been identified and linked to familial ALS (fALS). Although the precise mechanism(s) by which this diverse group of mutations causes fALS remains unclear, it generally is agreed that the ALSmutant SOD1 proteins are prone to misfold and that they acquire toxic properties as a consequence (1-3). Abnormal protein deposits are seen frequently in protein misfolding diseases, and SOD1-containing aggregates have been found consistently in the spinal cords of ALS transgenic mice and fALS patients (4, 5). Moreover, in ALS transgenic mice, these proteinaceous deposits frequently have been shown to have amyloid-like properties such as filamentous structures and the ability to bind thioflavin-S (6-8).A number of other proteins that have been linked to neurodegenerative diseases form amyloid fibrils. Such fibrils are elongated, unbranched, and highly ordered protein aggregates composed mainly of cross-β-sheets, with parallel or anti-parallel β-strands stacking perpendicular to the axis of fibril growth (9-13).Detergent-resistant aggregates isolated from the spinal cords of ALS transgenic mice contain full-length and metal-free human SOD1 (hSOD1) proteins, suggesting that it is full-length metal-free (apo) hSOD1 that acquires toxic properties in the disease mechanism (14). Moreover, as esta...

Arresting Amyloid with Coulomb’s Law: Acetylation of ALS-Linked SOD1 by Aspirin Impedes Aggregation

“…4, D-F, and 5, D-F). The typical diameter of unacetylated and acetylated fibrils was~15 to 20 nm, similar to previous studies (48).…”

Structural and kinetic analysis of protein-aggregate strains in vivo using binary epitope mapping

“…This suggests that mutant and wild-type hSOD1 might confer disease by the same mechanism: Human SOD1 seems to be an oversaturated protein with an intrinsic propensity to aggregate (13), and this tendency can be critically augmented by mutation. Consistently, mutant and wild-type hSOD1 show indistinguishable fibrillation behavior in vitro with kinetics determined by structural stability and net charge (14,15). The in vitro fibrillation occurs moreover by the recruitment of globally unfolded hSOD1 monomers, following exponential time courses controlled by fibril fragmentation (15).…”

“…The strain A and B patterns arising in vivo seem to contrast with the somewhat variable aggregate structures formed by the hSOD1 variants in vitro (14,16). One explanation would be that the CNS modulates the hSOD1 aggregation process by funneling toward uniform aggregate structures distinct from those that spontaneously form under simplified conditions in vitro.…”

“…An outstanding feature of hSOD1 aggregation, however, is a pronounced malleability, leading to different aggregate structures upon point mutation and altered experimental conditions (14,16). Like proteins in general, hSOD1 contains several "sticky" sequence regions, allowing a large number of competing aggregation pathways (17,18).…”

Aggregation-triggering segments of SOD1 fibril formation support a common pathway for familial and sporadic ALS

“…2, Left). The existence of several aggregation-prone segments in SOD1 unifies conflicting studies that find different regions from the protein participate in aggregate formation (19,20).…”