Mutations Enhance the Aggregation Propensity of the Alzheimer’s Aβ Peptide

Abstract

Aggregation of the amyloid β peptide (Aβ) plays a key role in the molecular etiology of Alzheimer's disease. Despite the importance of this process, the relationship between the sequence of Aβ and the propensity of the peptide to aggregate has not been fully elucidated. The sequence determinants of aggregation can be revealed by probing the ability of amino acid substitutions (mutations) to increase or decrease aggregation. Numerous mutations that decrease aggregation have been isolated by laboratory-based studies. In contrast, very few mutations that increase aggregation have been reported, and most of these were isolated from rare individuals with early onset Familial Alzheimer's disease. To augment the limited data set of clinically derived mutations, we developed an artificial genetic screen to isolate novel mutations that increase aggregation propensity. The screen relies on expression in E. coli of a fusion of Aβ to green fluorescent protein (GFP). In this fusion, the ability of the GFP reporter to fold and fluoresce is inversely correlated with the aggregation propensity of the Aβ sequence. Implementation of this screen enabled the isolation of 20 mutant versions of Aβ with amino acid substitutions at 17 positions in the 42-residue sequence of Aβ. Biophysical studies of synthetic peptides corresponding to sequences isolated by the screen confirm the increased aggregation propensity and amyloidogenic behavior of the mutants. The mutations were isolated using an unbiased screen that makes no assumptions about the sequence determinants of aggregation. Nonetheless, all 16 of the most aggregating mutants contain substitutions that reduce charge and/or increase hydrophobicity. These findings provide compelling evidence supporting the hypothesis that sequence hydrophobicity is a major determinant of Aβ aggregation.

The Alzheimer Disease Protective Mutation A2T Modulates Kinetic and Thermodynamic Properties of Amyloid-β (Aβ) Aggregation

“…Experimental evidence nevertheless suggests that N-terminal substitutions can affect A␤ aggregation (5,24,25,43). For instance, before being identified in the clinic, the A2V mutation itself was isolated as an aggregation-prone variant by screening mutant A␤40-GFP fusions in Escherichia coli (44).…”

“…1) are known to increase A␤ generation. Other mutations in the C-terminal part of A␤ (amino acids [43][44][45][46] shift the initial ⑀-cut by ␥-secretase from amino acids 50 -51 to amino acids 49 -50 and increase the relative amount of long, more aggregation-prone A␤ fragments versus shorter peptides, without increasing the total amount of A␤ (7)(8)(9). Other mutations, e.g.…”

Converting the Highly Amyloidogenic Human Calcitonin into a Powerful Fibril Inhibitor by Three-dimensional Structure Homology with a Non-amyloidogenic Analogue

“…In fact, in vitro fibril formation of globular proteins and of short peptides can be modulated by changes in the amino acid sequence (38,(65)(66)(67)(68). A "structural" view of fibrillation suggests that a way to prevent it is to stabilize an ␣/␤ discordant helix (i.e.…”

The Osaka FAD Mutation E22Δ Leads to the Formation of a Previously Unknown Type of Amyloid β Fibrils and Modulates Aβ Neurotoxicity

“…22 A fraction of 5-9% impurities may thus have prevented the aggregation of Aβ1-40 E22Δ and Aβ1-42 E22Δ in the study of Tomiyama et al 5 Our results are in agreement with previous studies on charge alterations within the Aβ sequence, where a change or a loss of charge at position 22 in Aβ enhanced both the aggregation propensity and the toxicity of the corresponding mutant peptides. 34,35 Aβ variants corresponding to the FAD mutations Dutch (E22Q), Italian (E22K), and Arctic (E22G) aggregated much faster than wt Aβ. 12,36,37 We found that the Aβ1-42 E22Δ showed an increased tendency of β-sheet conformation and aggregated faster than Aβ1-42 wt.…”