Exposure of hydrophobic surfaces initiates aggregation of diverse ALS-causing superoxide dismutase-1 mutants.
Bottom Line: The remarkable diversity of the effects of these mutations on SOD1 properties has suggested that they promote aggregation by a variety of mechanisms.Our results uncover the biochemical nature of the misfolded aggregation-prone intermediate and reconcile the seemingly diverse effects of ALS-causing mutations into a unifying mechanism.Furthermore, the method we describe here will be useful for investigating and interfering with aggregation of various proteins and thereby provide insight into the molecular mechanisms underlying many neurodegenerative diseases.
Affiliation: MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.Show MeSH
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Mentions: In all conditions examined using two specific SOD1 mutants, we observed a correlation between exposure of hydrophobic surfaces and aggregation. To determine whether increased exposure of hydrophobic surfaces correlates with aggregation of diverse SOD1 mutants, we monitored aggregation and exposure of hydrophobic surfaces of the representative set of 12 mutants. As-purified SOD1WT and mutant proteins were exposed to 20% TFE, a condition that was found to elicit aggregation of the diverse SOD1 mutants, but not the wild-type protein. This treatment converted the soluble MBR mutants into aggregates in 20 minutes and was also efficient to provoke aggregation of the WTL mutants (Supplementary Fig. 6a). Exposure of surface hydrophobicity was monitored by Sypro Orange fluorescence and aggregation monitored both by DLS and ThT fluorescence (Fig. 6a and b). ThT fluorescence indicated that aggregates have an amyloid component. Analysis of Congo Red binding confirmed this (Supplementary Fig. 6b). We found exposure of hydrophobicity and ordered assembly of SOD1 aggregates, monitored by these two independent methods, were strongly correlated. The strength of the correlation between exposure of hydrophobic surfaces and aggregation is attested by the very high linear correlation coefficient (Fig. 6a, R = 0.95 and b, R = 0.84). This revealed that exposure of hydrophobicity is a common feature that provokes aggregation of diverse SOD1 mutants.
Affiliation: MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.