Abstract
The acylation of lysine residues in superoxide dismutase-1 (SOD1) has been previously shown to decrease its rate of nucleation and elongation into amyloid-like fibrils linked to amyotrophic lateral sclerosis. The chemical mechanism underlying this effect is unclear, i.e. hydrophobic/steric effects versus electrostatic effects. Moreover, the degree to which the acylation might alter the prion-like seeding of SOD1 in vivo has not been addressed. Here, we acylated a fraction of lysine residues in SOD1 with groups of variable hydrophobicity, charge, and conformational entropy. The effect of each acyl group on the rate of SOD1 fibril nucleation and elongation were quantified in vitro with thioflavin-T (ThT) fluorescence, and we performed 594 iterate aggregation assays to obtain statistically significant rates. The effect of the lysine acylation on the prion-like seeding of SOD1 was assayed in spinal cord extracts of transgenic mice expressing a G85R SOD1-yellow fluorescent protein construct. Acyl groups with >2 carboxylic acids diminished self-assembly into ThT-positive fibrils and instead promoted the self-assembly of ThT-negative fibrils and amorphous complexes. The addition of ThT-negative, acylated SOD1 fibrils to organotypic spinal cord failed to produce the SOD1 inclusion pathology that typically results from the addition of ThT-positive SOD1 fibrils. These results suggest that chemically increasing the net negative surface charge of SOD1 via acylation can block the prion-like propagation of oligomeric SOD1 in spinal cord.
Highlights
The acylation of lysine residues in superoxide dismutase-1 (SOD1) has been previously shown to decrease its rate of nucleation and elongation into amyloid-like fibrils linked to amyotrophic lateral sclerosis
Ϫ2.2 a Average stoichiometry of the acylated protein was calculated as a weighted average of integrated mass spectra. b Ratios for all kinetic parameters were calculated as mod//unmod, kmod/kunmod, and ThTmod/ThTunmod, with p values calculated with respect to unmodified SOD1 using an unpaired Student’s t test at 95% confidence interval (p Ͻ 0.05). c Hazard ratios and their corresponding p values were calculated from the statistical comparison between Kaplan–Meier plots of each modified SOD1 and its corresponding unmodified protein, using the log-rank (Mantel–Cox) algorithm at 95% confidence interval (p Ͻ 0.05). d Calculated via integration of deconvoluted mass spectra. e The values of ⌬Tm are calculated as Tm(unmod) Ϫ Tm(acylated)
This paper demonstrates that attachment of negatively charged acyl groups to a fraction of lysine residues in SOD1 can inhibit its aggregation into ThT-positive fibrils that lack seeding activity in organotypic spinal cord, while promoting the formation of ThT-negative fibrils and amorphous complexes that lack seeding activity in organotypic spinal cord
Summary
The natural and unnatural acyl modifications examined in this study increase the magnitude of net negative charge of SOD1 by Ϫ1 to Ϫ4 formal units; hydrophobicity of the lysine⑀-NH3ϩ is increased from log P Ϸ ϩ1.0 to log P Ϸ Ϫ2.0 (Fig. 2). The percentage of SOD1 that remained unacylated after addition of anhydrides ranged from 10.7% in tricarboxycyclobutylated samples to 40.9% in tricarboxybiphenylated samples; unacetylated SOD1 ranged from 20.8 to 36.2% for all other types of acyl groups (Table 1). These stoichiometries were confirmed with capillary electrophoresis (CE), which detects proteins by absorbance at 214 nm (Fig. 3, c and d). Trypsin digestion and tandem mass spectrometry resulted in between 98 and 100% sequence coverage and suggested that lysine residues were semirandomly acylated by each anhydride Kinetic parameters of in vitro fibrilization of WT apo-SOD1 (100 mM TCEP, 37 °C, pH 7.4) before and after acylation The data are shown as means Ϯ S.D
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