Fibrillation precursor of superoxide dismutase 1 revealed by gradual tuning of the protein-folding equilibrium

Abstract

Although superoxide dismutase 1 (SOD1) stands out as a relatively soluble protein in vitro, it can be made to fibrillate by mechanical agitation. The mechanism of this fibrillation process is yet poorly understood, but attains considerable interest due to SOD1's involvement in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). In this study, we map out the apoSOD1 fibrillation process from how it competes with the global folding events at increasing concentrations of urea: We determine how the fibrillation lag time (τ lag ) and maximum growth rate (ν max ) depend on gradual titration of the folding equilibrium, from the native to the unfolded state. The results show that the agitation-induced fibrillation of apoSOD1 uses globally unfolded precursors and relies on fragmentation-assisted growth. Mutational screening and fibrillation m-values (∂ log τ lag ∕∂½urea and ∂ log ν max ∕∂½urea) indicate moreover that the fibrillation pathway proceeds via a diffusely bound transient complex that responds to the global physiochemical properties of the SOD1 sequence. Fibrillation of apoSOD1, as it bifurcates from the denatured ensemble, seems thus mechanistically analogous to that of disordered peptides, save the competing folding transition to the native state. Finally, we examine by comparison with in vivo data to what extent this mode of fibrillation, originating from selective amplification of mechanically brittle aggregates by sample agitation, captures the mechanism of pathological SOD1 aggregation in ALS.I ntracellular aggregation of the homodimeric enzyme superoxide dismutase (SOD1) is a key pathological hallmark of amyotrophic lateral sclerosis (ALS). Despite this coupling with protein-aggregation disease, SOD1 stands out as a relatively soluble protein in vitro: Mutations that are destabilized to the extent that they fail to fold can be left for weeks in physiological buffer without aggregating. Studies of SOD1 aggregation need then to involve certain stimuli that prompts the process to occur (1). One such stimulus is mechanical agitation. Like many other proteins, SOD1 can be driven to form amyloid-like fibrils by shaking or stirring the sample (2-4). Although these experiments may at first seem artificial, they have come to play a key role in our understanding of the aggregation mechanism: Protein aggregation is a high-dimensional process and, as such, its predicted outcome relies on knowledge about all competing pathways (5). In the case of SOD1, this high dimensionality is underlined by the observation that the structures of the fibrillar aggregates are polymorphic and vary with mutation (6). Competing aggregation pathways are also indicated at macroscopic level where the morphology of the SOD1 aggregates can be changed from filamentous to branched protofibrillar or amorphous by replacing the mechanical agitation with incubation in organic solvent (7), addition of lipids (8), or allowing oxidative cross-linking of the protein's solvent accessible cysteines (9). Non-amyloid aggregates can also be...

SOD1 aggregation in ALS mice shows simplistic test tube behavior

“…2B). The result matches within error data from urea destabilization (7). Altogether, these observations show that the aggregation mechanism of SOD1 in vitro is robust and largely independent of how the protein is destabilized.…”

“…Effective Concentration of Aggregation-Competent Material. Unlike disordered peptides, globular proteins, like SOD1, need to unfold to aggregate (7). Thus, at any given moment, only a fraction ( f) of the protein molecules are aggregation-competent, whereas the rest (1 − f) remain folded and soluble (Fig.…”

“…A reductionist view, however, would be that analogous collapse of the human proteostasis occurs as a result of an abnormally age-declined or otherwise compromised housekeeping machinery, leaving SOD1 wt as an inherently oversaturated protein (52) to fall through (sporadic or familial SOD1 wt ALS). Alternatively, neurons expressing mutant SOD1 with elevated levels of immature apo species or decreased repulsive charge (7,35) raise the aggregate load to a point where collapse is bound to occur, even with the normally aging housekeeping system (familial SOD1 mutant ALS). If so, the SOD1 aggregate levels in the spinal cord should, in both cases, display a 40-to 60-y lag followed by a sudden exponential multiplication, leading to disease onset.…”

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).…”

Global structural motions from the strain of a single hydrogen bond

“…In contrast to the high-energy intermediates of β2-microglobulin and lysozyme, however, we observe here that the high-energy state HS is not on the pathway to the unfolded state, but is more compact than the solution ground state (Table 1). Together with the finding that destabilization of the apo monomer with urea accelerates the fibrillation kinetics in vitro (37), this puts precise constraints on the aggregation mechanism: the fibrillation precursor needs to be more expanded than the folded ground state, which excludes the involvement of the high-energy state HS. Instead, the precursor for apoSOD fibrillation appears to be the globally denatured state (37), consistent with an observed maximum of the fibrillation kinetics above 5 M urea, at which the occupancy of D is nearly 1 and the occupancy of the high-energy state has decreased from 0.01 to less than 10 −4.7 (Table 1, SI Text, and Fig.…”