Mutations in Cu͞Zn superoxide dismutase (SOD) are associated with the fatal neurodegenerative disorder amyotrophic lateral sclerosis (ALS). There is considerable evidence that mutant SOD has a gain of toxic function; however, the mechanism of this toxicity is not known. We report here that purified SOD forms aggregates in vitro under destabilizing solution conditions by a process involving a transition from small amorphous species to fibrils. The assembly process and the tinctorial and structural properties of the in vitro aggregates resemble those for aggregates observed in vivo. Furthermore, the familial ALS SOD mutations A4V, G93A, G93R, and E100G decrease protein stability, which correlates with an increase in the propensity of the mutants to form aggregates. These mutations also increase the rate of protein unfolding. Our results suggest three possible mechanisms for the increase in aggregation: (i) an increase in the equilibrium population of unfolded or of partially unfolded states, (ii) an increase in the rate of unfolding, and (iii) a decrease in the rate of folding. Our data support the hypothesis that the gain of toxic function for many different familial ALS-associated mutant SODs is a consequence of protein destabilization, which leads to an increase in the formation of cytotoxic protein aggregates.

Despite the widespread use of sonication in medicine, industry, and research, the effects of sonication on proteins remain poorly characterized. We report that sonication of a range of structurally diverse proteins results in the formation of aggregates that have similarities to amyloid aggregates. The formation of amyloid is associated with, and has been implicated in, causing of a wide range of protein conformational disorders including Alzheimer's disease, Huntington's disease, Parkinson's disease, and prion diseases. The aggregates cause large enhancements in fluorescence of the dye thioflavin T, exhibit green-gold birefringence upon binding the dye Congo red, and cause a red-shift in the absorbance spectrum of Congo red. In addition, circular dichroism reveals that sonication-induced aggregates have high ␤-content, and proteins with significant native ␣-helical structure show increased ␤-structure in the aggregates. Ultrastructural analysis by electron microscopy reveals a range of morphologies for the sonication-induced aggregates, including fibrils with diameters of 5-20 nm. The addition of preformed aggregates to unsonicated protein solutions results in accelerated and enhanced formation of additional aggregates upon heating. The dye-binding and structural characteristics, as well as the ability of the sonication-induced aggregates to seed the formation of new aggregates are all similar to the properties of amyloid. These results have important implications for the use of sonication in food, biotechnological and medical applications, and for research on protein aggregation and conformational disorders.

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Protein aggregation is a hallmark of many diseases, including amyotrophic lateral sclerosis (ALS), where aggregation of Cu/Zn superoxide dismutase (SOD1) is implicated in causing neurodegeneration. Recent studies have suggested that destabilization and aggregation of the most immature form of SOD1, the disulfide-reduced, unmetallated (apo) protein is particularly important in causing ALS. We report herein in depth analyses of the effects of chemically and structurally diverse ALS-associated mutations on the stability and aggregation of reduced apo SOD1. In contrast with previous studies, we find that various reduced apo SOD1 mutants undergo highly reversible thermal denaturation with little aggregation, enabling quantitative thermodynamic stability analyses. In the absence of ALS-associated mutations, reduced apo SOD1 is marginally stable but predominantly folded. Mutations generally result in slight decreases to substantial increases in the fraction of unfolded protein. Calorimetry, ultracentrifugation, and light scattering show that all mutations enhance aggregation propensity, with the effects varying widely, from subtle increases in most cases, to pronounced formation of 40–100 nm soluble aggregates by A4V, a mutation that is associated with particularly short disease duration. Interestingly, although there is a correlation between observed aggregation and stability, there is minimal to no correlation between observed aggregation, predicted aggregation propensity, and disease characteristics. These findings suggest that reduced apo SOD1 does not play a dominant role in modulating disease. Rather, additional and/or multiple forms of SOD1 and additional biophysical and biological factors are needed to account for the toxicity of mutant SOD1 in ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease involving cytotoxic conformations of Cu, Zn superoxide dismutase (SOD1). A major challenge in understanding ALS disease pathology has been the identification and atomic-level characterization of these conformers. Here, we use a combination of NMR methods to detect four distinct sparsely populated and transiently formed thermally accessible conformers in equilibrium with the native state of immature SOD1 (apoSOD12SH). Structural models of two of these establish that they possess features present in the mature dimeric protein. In contrast, the other two are non-native oligomers in which the native dimer interface and the electrostatic loop mediate the formation of aberrant intermolecular interactions. Our results show that apoSOD12SH has a rugged free energy landscape that codes for distinct kinetic pathways leading to either maturation or non-native association and provide a starting point for a detailed atomic-level understanding of the mechanisms of SOD1 oligomerization.DOI: http://dx.doi.org/10.7554/eLife.07296.001