Reaction network starting from monomer mixtures of Aβ40 and Aβ42. Interaction at the level of primary nucleation only accelerates Aβ40 fibril formation. Separate fibrils form as secondary nucleation and elongation are highly specific.
Mapping energy landscapes has proved to be a powerful tool for studying reaction mechanisms. Many complex biomolecular assembly processes, however, have remained challenging to access using this approach, including the aggregation of peptides and proteins into amyloid fibrils implicated in various disorders. Here we generalize the strategy used to probe energy landscapes in protein folding to determine the activation energies and entropies that characterise each of the molecular steps in the aggregation of the amyloid-β peptide (Aβ42), which is associated with Alzheimer’s disease. Our results reveal that interactions between monomeric Aβ and amyloid fibrils during fibril-dependent nucleation fundamentally reverse the thermodynamic signature of this process relative to primary nucleation, even though both processes generate aggregates from soluble peptides. By mapping the energetic and entropic contributions along the reactive trajectories, we show that the catalytic efficiency of Aβ42 fibril surfaces results from the enthalpic stabilisation of adsorbing peptides in conformations amenable to nucleation, driving a dramatic lowering of the activation energy barrier for nucleation.
The aggregation of the amyloid beta peptide, Aβ42, implicated in Alzheimer's disease, is characterized by a lag phase followed by a rapid growth phase. Conventional methods to study this reaction are not sensitive to events taking place early in the lag phase promoting the assumption that only monomeric or oligomeric species are present at early stages and that the lag time is defined by the primary nucleation rate only. Here we exploit the high sensitivity of chemical chain reactions to the reagent composition to develop an assay which improves by 2 orders of magnitude the detection limit of conventional bulk techniques and allows the concentration of fibrillar Aβ42 propagons to be detected and quantified even during the lag time. The method relies on the chain reaction multiplication of a small number of initial fibrils by secondary nucleation on the fibril surface in the presence of monomeric peptides, allowing the quantification of the number of initial propagons by comparing the multiplication reaction kinetics with controlled seeding data. The quantitative results of the chain reaction assay are confirmed by qualitative transmission electron microscopy analysis. The results demonstrate the nonlinearity of the aggregation process which involves both primary and secondary nucleation events even at the early stages of the reaction during the lag-phase.
The human molecular chaperone DNAJB6, an oligomeric protein with a conserved S/T-rich region, is an efficient suppressor of amyloid fibril formation by highly aggregation-prone peptides such as the Aβ and polyQ peptides associated with Alzheimer's and Huntington's disease, respectively. We previously showed that DNAJB6 can inhibit the processes through which amyloid fibrils are formed via strong interactions with aggregated forms of Aβ42 that become sequestered. Here we report that the concentration-dependent capability of DNAJB6 to suppress fibril formation in thioflavin T fluorescence assays decreases progressively with an increasing number of S/T substitutions, with an almost complete loss of suppression when 18 S/T residues are substituted. The kinetics of primary nucleation in particular are affected. No detectable changes in the structure are caused by the substitutions. Also, the level of binding of DNAJB6 to Aβ42 decreases with the S/T substitutions, as determined by surface plasmon resonance and microscale thermophoresis. The aggregation process monitored using nuclear magnetic resonance spectroscopy showed that DNAJB6, in contrast to a mutational variant with 18 S/T residues substituted, can keep monomeric Aβ42 soluble for an extended time. The inhibition of the primary nucleation is likely to depend on hydroxyl groups in side chains of the S/T residues, and hydrogen bonding with Aβ42 is one plausible molecular mechanism, although other possibilities cannot be excluded. The loss of the ability to suppress fibril formation upon S/T to A substitution was previously observed also for polyQ peptides, suggesting that the S/T residues in the DNAJB6-like chaperones have a general ability to inhibit amyloid fibril formation by different aggregation-prone peptides.
Aggregation of the amyloid β-protein (Aβ) is believed to be involved in Alzheimer's disease pathogenesis. Here we have investigated the importance of the aromatic rings at positions 19 and 20 for the aggregation rate and mechanism by substituting phenylalanine with leucine. Aggregation kinetics were monitored as a function of time and peptide concentration by thioflavin T (ThT) fluorescence, the aggregation equilibrium by sedimentation assay, structural changes using circular dichroism spectroscopy and the presence of fibrillar material was detected with cryotransmission electron microscopy. All peptides convert from monomer to amyloid fibrils in a concentration-dependent manner. Substituting F19 with leucine results in a peptide that aggregates significantly slower than the wild type, while substitution of F20 produces a peptide that aggregates faster. The effects of the two substitutions are additive, since simultaneous substitution of F19 and F20 produces a peptide with aggregation kinetics intermediate between F19L and F20L. Our results suggest that the aromatic side-chain of F19 favors nucleation of the aggregation process and may be an important target for therapeutic intervention.
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