N-Terminal Extensions Retard Aβ42 Fibril Formation but Allow Cross-Seeding and Coaggregation with Aβ42

Abstract

Amyloid β-protein (Aβ) sequence length variants with varying aggregation propensity coexist in vivo, where coaggregation and cross-catalysis phenomena may affect the aggregation process. Until recently, naturally occurring amyloid β-protein (Aβ) variants were believed to begin at or after the canonical β-secretase cleavage site within the amyloid β-protein precursor. However, N-terminally extended forms of Aβ (NTE-Aβ) were recently discovered and may contribute to Alzheimer's disease. Here, we have used thioflavin T fluorescence to study the aggregation kinetics of Aβ42 variants with N-terminal extensions of 5–40 residues, and transmission electron microscopy to analyze the end states. We find that all variants form amyloid fibrils of similar morphology as Aβ42, but the half-time of aggregation (t1/2) increases exponentially with extension length. Monte Carlo simulations of model peptides suggest that the retardation is due to an underlying general physicochemical effect involving reduced frequency of productive molecular encounters. Indeed, global kinetic analyses reveal that NTE-Aβ42s form fibrils via the same mechanism as Aβ42, but all microscopic rate constants (primary and secondary nucleation, elongation) are reduced for the N-terminally extended variants. Still, Aβ42 and NTE-Aβ42 coaggregate to form mixed fibrils and fibrils of either Aβ42 or NTE-Aβ42 catalyze aggregation of all monomers. NTE-Aβ42 monomers display reduced aggregation rate with all kinds of seeds implying that extended termini interfere with the ability of monomers to nucleate or elongate. Cross-seeding or coaggregation may therefore represent an important contribution in the in vivo formation of assemblies believed to be important in disease.

Solid‐Phase Synthesis and Characterization of N‐Terminally Elongated Aβ−3–x‐Peptides

“…From the comparison of Aβ −3– x with Aβ 1– x we suggest that N‐terminal elongation increases the propensity for aggregation, although the monomer incorporation rate to preformed aggregates does not seem to be significantly affected. This is in apparent contrast with a very recent study, in which the impact of N‐terminal elongation, ranging from 5–40 amino acids in length, on the aggregation kinetics of recombinant variants of human Aβ 42 was investigated . All of the N‐terminally extended forms of Aβ 42 under investigation were shown to be able to form amyloid fibrils similar to those formed from Aβ 1–42 , but with lower rates.…”

“…This is in apparent contrast with av ery recent study,i nw hich the impact of N-terminal elongation, ranging from 5-40 amino acids in length, on the aggregation kinetics of recombinant variants of human Ab 42 was investigated. [31] All of the N-terminallye xtended forms of Ab 42 under investigationw ere shown to be able to form amyloid fibrils similar to those formed from Ab 1-42 ,b ut with lower rates. The apparent discrepancyb etween these two studies may result from differences in experimental procedures such as solubilization of Ab peptidep rior to aggregation assays or the different lengths of the N-terminalextensions.…”

Evidence for a central role of PrP helix 2 in the nucleation of amyloid fibrils

“…Alternatively, the H3 region might compete with an intermolecular contact between the amyloidogenic regions (i.e., the H2 region); accordingly, the H3 deletion would increase the probability of the productive contact leading to the nucleus formation. Szczepankiewicz et al (65) recently proposed this mechanism based on their observation that N-terminal extensions of amyloid-b with nonamyloidogenic sequences significantly impedes the nucleation of amyloid fibrils. One might also assume that the H3 region forms a transient nonamyloid structure that destabilizes the overall structure of the nuclei.…”

AFM-Based Single Molecule Techniques: Unraveling the Amyloid Pathogenic Species

“…In other words, once a small but critical concentration of Aβ42 aggregates has been generated through primary nucleation of monomers, surface-catalyzed secondary nucleation becomes the dominant process where the surface of the existing fibrils serve as catalytic sites for the generation of toxic oligomeric species [54, 57]. Furthermore, the role of intrinsic and extrinsic factors on the aggregation process of Aβ42 has been partly unveiled and a great effort has been focused on drug development against Aβ42 aggregation, which has proven to be very difficult [100, 101]. In agreement, no compound has cleared clinical trials and entered clinical use [102, 103].…”