Structural Analysis and Aggregation Propensity of Pyroglutamate Aβ(3-40) in Aqueous Trifluoroethanol

Abstract

A hallmark of Alzheimer’s disease (AD) is the accumulation of extracellular amyloid-β (Aβ) plaques in the brains of patients. N-terminally truncated pyroglutamate-modified Aβ (pEAβ) has been described as a major compound of Aβ species in senile plaques. pEAβ is more resistant to degradation, shows higher toxicity and has increased aggregation propensity and β-sheet stabilization compared to non-modified Aβ. Here we characterized recombinant pEAβ(3–40) in aqueous trifluoroethanol (TFE) solution regarding its aggregation propensity and structural changes in comparison to its non-pyroglutamate-modified variant Aβ(1–40). Secondary structure analysis by circular dichroism spectroscopy suggests that pEAβ(3–40) shows an increased tendency to form β-sheet-rich structures in 20% TFE containing solutions where Aβ(1–40) forms α-helices. Aggregation kinetics of pEAβ(3–40) in the presence of 20% TFE monitored by thioflavin-T (ThT) assay showed a typical sigmoidal aggregation in contrast to Aβ(1–40), which lacks ThT positive structures under the same conditions. Transmission electron microscopy confirms that pEAβ(3–40) aggregated to large fibrils and high molecular weight aggregates in spite of the presence of the helix stabilizing co-solvent TFE. High resolution NMR spectroscopy of recombinantly produced and uniformly isotope labeled [U-15N]-pEAβ(3–40) in TFE containing solutions indicates that the pyroglutamate formation affects significantly the N-terminal region, which in turn leads to decreased monomer stability and increased aggregation propensity.

Pyroglutamate-Modified Amyloid-β(3–42) Shows α-Helical Intermediates before Amyloid Formation

“…Under conditions where the thermodynamic equilibrium is not reached, helical Ab (1-42) is more stabilized by TFE than pEAb , as pEAb shows helix-to-sheet conversion at lower TFE concentrations. This is consistent with the behavior of the C-terminally shortened isoforms pEAb and Ab (1-40) (23).…”

“…The results indicated that pyroglutamate-modified Ab has a decreased helix propensity along with increased hydrophobicity and faster aggregation kinetics, even in TFE-water mixtures. We have previously shown that pEAb forms b-sheet-containing structures under conditions where the non-modified isoform Ab (1-40) forms a-helices (23). Here, we extended these studies by investigating monomeric as well as oligomeric pEAb before aggregation.…”

Pyroglutamate‐Modified Amyloid β (11‐ 40) Fibrils Are More Toxic than Wildtype Fibrils but Structurally Very Similar

“…The pyroglutamate residue 11 was 13 C/ 15 N labeled following the procedure described in ref. . The peptides were solubilized in 50 m m Tris buffer (100 m m NaCl, 0.01 % NaN 3 , pH 8) at a concentration of 0.1 mg mL −1 .…”

Unmodified and pyroglutamylated amyloid β peptides form hypertoxic hetero‐oligomers of unique secondary structure

“…AbpE was shown to have a higher tendency toward formation of b-sheet aggregates, possibly promoting aggregation of Ab by a seeding mechanism [21,[40][41][42][43] and to be hypertoxic to neuronal cells [18][19][20][21]. Ab pE3-40 in trifluoroethanol/water environment had an increased tendency toward bsheet formation and fibrillization compared to Ab 1-40 [44,45], as well as higher susceptibility to mechanical stress and fragmentation of the fibrils [46]. These reports have been challenged by other studies, arguing that AbpE assemblies have similar content of b-sheet structure, are more resistant to fibrillogenesis [47][48][49][50], and exert cytotoxic effect similar to that of unmodified Ab [47,50,51].…”