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N-Terminal Extensions Retard A?42 Fibril Formation but Allow Cross-Seeding and Coaggregation with A?42.

ABSTRACT: Amyloid ?-protein (A?) 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 ?-protein (A?) variants were believed to begin at or after the canonical ?-secretase cleavage site within the amyloid ?-protein precursor. However, N-terminally extended forms of A? (NTE-A?) were recently discovered and may contribute to Alzheimer's disease. Here, we have used thioflavin T fluorescence to study the aggregation kinetics of A?42 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 A?42, 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-A?42s form fibrils via the same mechanism as A?42, but all microscopic rate constants (primary and secondary nucleation, elongation) are reduced for the N-terminally extended variants. Still, A?42 and NTE-A?42 coaggregate to form mixed fibrils and fibrils of either A?42 or NTE-A?42 catalyze aggregation of all monomers. NTE-A?42 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.

SUBMITTER: Szczepankiewicz O

PROVIDER: S-EPMC5412961 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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

Szczepankiewicz Olga O Linse Björn B Meisl Georg G Thulin Eva E Frohm Birgitta B Sala Frigerio Carlo C Colvin Michael T MT Jacavone Angela C AC Griffin Robert G RG Knowles Tuomas T Walsh Dominic M DM Linse Sara S

Journal of the American Chemical Society 20151117 46

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 h ...[more]

PMID: 26535489

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Project description:The difficulty in identifying the toxic agents in all amyloid-related diseases is likely due to the complicated kinetics and thermodynamics of the nucleation process and subsequent fibril formation. The slow progression of these diseases suggests that the formation, incorporation, and/or action of toxic agents are possibly rate limiting. Candidate toxic agents include precursors (some at very low concentrations), also called oligomers and protofibrils, and the fibrils. Here, we investigate the kinetic and thermodynamic behavior of human insulin oligomers (imaged by cryo-EM) under fibril-forming conditions (pH 1.6 and 65 degrees C) by probing the reaction pathway to insulin fibril formation using two different types of experiments-cooling and seeding-and confirm the validity of the nucleation model and its effect on fibril growth. The results from both the cooling and seeding studies confirm the existence of a time-changing oligomer reaction process prior to fibril formation that likely involves a rate-limiting nucleation process followed by structural rearrangements of intermediates (into beta-sheet rich entities) to form oligomers that then form fibrils. The latter structural rearrangement step occurs even in the absence of nuclei (i.e., with added heterologous seeds). Nuclei are formed at the fibrillation conditions (pH 1.6 and 65 degrees C) but are also continuously formed during cooling at pH 1.6 and 25 degrees C. Within the time-scale of the experiments, only after increasing the temperature to 65 degrees C are the trapped insulin nuclei and resultant structures able to induce the structural rearrangement step and overcome the energy barrier to form fibrils. This delay in fibrillation and accumulation of nuclei at low temperature (25 degrees C) result in a decrease in the mean length of the fibers when placed at 65 degrees C. Fits of an empirical model to the data provide quantitative measures of the delay in the lag-time during the nucleation process and subsequent reduction in fibril growth rate resulting from the cooling. Also, the seeding experiments, within the time-scale of the measurements, demonstrate that fibers can initiate fast fibrillation with dissolved insulin (fresh or taken during the lag-period) but not with other fibers. Qualitatively this is explained with a conjectual free-energy space plot.

Dataset Information

N-Terminal Extensions Retard A?42 Fibril Formation but Allow Cross-Seeding and Coaggregation with A?42.

Publications

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

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