Cu/Zn Superoxide Dismutase Forms Amyloid Fibrils under Near-Physiological Quiescent Conditions: The Roles of Disulfide Bonds and Effects of Denaturant

Abstract

Cu/Zn superoxide dismutase (SOD1) forms intracellular aggregates that are pathological indicators of amyotrophic lateral sclerosis. A large body of research indicates that the entry point to aggregate formation is a monomeric, metal-ion free (apo), and disulfide-reduced species. Fibril formation by SOD1 in vitro has typically been reported only for harsh solvent conditions or mechanical agitation. Here we show that monomeric apo-SOD1 in the disulfide-reduced state forms fibrillar aggregates under near-physiological quiescent conditions. Monomeric apo-SOD1 with an intact intramolecular disulfide bond is highly resistant to aggregation under the same conditions. A cysteine-free variant of SOD1 exhibits fibrillization behavior and fibril morphology identical to those of disulfide-reduced SOD1, firmly establishing that intermolecular disulfide bonds or intramolecular disulfide shuffling are not required for aggregation and fibril formation. The decreased lag time for fibril formation resulting from reduction of the intramolecular disulfide bond thus primarily reflects the decreased stability of the folded state relative to partially unfolded states, rather than an active role of free sulfhydryl groups in mediating aggregation. Addition of urea to increase the amount of fully unfolded SOD1 increases the lag time for fibril formation, indicating that the population of this species does not dominate over other factors in determining the onset of aggregation. Our results contrast with previous results obtained for agitated samples, in which case amyloid formation was accelerated by denaturant. We reconcile these observations by suggesting that denaturants destabilize monomeric and aggregated species to different extents and thus affect nucleation and growth.

Large SOD1 aggregates, unlike trimeric SOD1, do not impact cell viability in a model of amyotrophic lateral sclerosis

“…Upon extended incubation, misfolded monomers and aggregates became the dominant species. In agreement with previous findings, our results suggest the monomeric species forms the available pool for further oligomerization and fibrillation of SOD1, and that the trimeric species represents an intermediate, metastable state (9,25).…”

Adsorption of unfolded Cu/Zn superoxide dismutase onto hydrophobic surfaces catalyzes its formation of amyloid fibrils

“…The resulting monomeric, disulfide reduced and metal-ion free (apo) state is the most immature species of SOD1 expected in the cell and it could conceivably accumulate under various conditions of cell stress. A large number of studies have implicated this species as the precursor of aggregated forms (Hough et al, 2004;Khare et al, 2004;Rakhit et al, 2004;Lang et al, 2012;Khan et al, 2017). In keeping with the general view of amyloid formation Dobson, 2006, 2009) it is believed that a partially or globally unfolded conformation of SOD1 is the starting point for aggregation.…”

“…week (Khan et al, 2017). In our previous study we monitored fibril formation using PEGylated microplates, and found that increasing denaturant concentration delayed the onset of fibril formation.…”

“…First, the greater hydrophobic surface, contributed by the air-water interface and teflon-coated stirrers, present in agitated samples is expected to favor adsorption of unfolded protein with exposed hydrophobic patches, which increase with increasing denaturant concentration, leading to surface-catalyzed nucleation of fibril formation (Morinaga et al, 2010;Pronchik et al, 2010). By contrast, in the case of non-agitated experiments using PEGylated sample containers, denaturants apparently destabilize aggregated species, such as oligomers and fibrils, and this effect is more important than the concomitant destabilization of the native monomeric state, leading to a delayed onset of fibril formation (Khan et al, 2017). In the presence of extraneous hydrophobic surfaces the destabilizing effect of denaturants on aggregated species is apparently reduced significantly.…”

“…The structure of pwt-SOD1 shows no significant deviations from wild-type SOD1 (Hörnberg et al, 2007). We also used a cysteine-free mutant (SOD1 DC ) that includes the C57S and C146S mutations on the background of the pwt-SOD1 template (Khan et al, 2017). Furthermore, for comparison with previous studies (Danielsson et al, 2011) we also included the loop-less variant (SOD1 LL ), in which both the metal-binding loop (residues 49-81, loop IV) and electrostatic loop (residues 124-139, loop VII) are replaced by Gly-Ala-Gly tripeptide linkers.…”