The role of shear forces in primary and secondary nucleation of amyloid fibrils.

Shear forces affect self-assembly processes ranging from crystallization to fiber formation. Here, the effect of mild agitation on amyloid fibril formation was explored for four peptides and investigated in detail for A[Formula: see text]42, which is associated with Alzheimer's disease. To gain mechanistic insights into the effect of mild agitation, nonseeded and seeded aggregation reactions were set up at various peptide concentrations with and without an inhibitor. First, an effect on fibril fragmentation was excluded by comparing the monomer-concentration dependence of aggregation kinetics under idle and agitated conditions. Second, using a secondary nucleation inhibitor, Brichos, the agitation effect on primary nucleation was decoupled from secondary nucleation. Third, an effect on secondary nucleation was established in the absence of inhibitor. Fourth, an effect on elongation was excluded by comparing the seeding potency of fibrils formed under idle or agitated conditions. We find that both primary and secondary nucleation steps are accelerated by gentle agitation. The increased shear forces facilitate both the detachment of newly formed aggregates from catalytic surfaces and the rate at which molecules are transported in the bulk solution to encounter nucleation sites on the fibril and other surfaces. Ultrastructural evidence obtained with cryogenic transmission electron microscopy and free-flow electrophoresis in microfluidics devices imply that agitation speeds up the detachment of nucleated species from the fibril surface. Our findings shed light on the aggregation mechanism and the role of detachment for efficient secondary nucleation. The results inform on how to modulate the relative importance of different microscopic steps in drug discovery and investigations.

The Ability of DNAJB6b to Suppress Amyloid Formation Depends on the Chaperone Aggregation State.

For many chaperones, a propensity to self-assemble correlates with function. The highly efficient amyloid suppressing chaperone DNAJB6b has been reported to oligomerize. A key question is whether the DNAJB6b self-assemblies or their subunits are active units in the suppression of amyloid formation. Here, we address this question using a nonmodified chaperone. We use the well-established aggregation kinetics of the amyloid ß 42 peptide (Aß42) as a readout of the amyloid suppression efficiency. The experimental setup relies on the slow dissociation of DNAJB6b assemblies upon dilution. We find that the dissociation of the chaperone assemblies correlates with its ability to suppress fibril formation. Thus, the data show that the subunits of DNAJB6b assemblies rather than the large oligomers are the active forms in amyloid suppression. Our results provide insights into how DNAJB6b operates as a chaperone and illustrate the importance of established assembly equilibria and dissociation rates for the design of kinetic experiments.

A label-free high-throughput protein solubility assay and its application to Aß40.

A major hallmark of Alzheimer's disease is the accumulation of aggregated amyloid ß peptide (Aß) in the brain. Here we develop a solubility assay for proteins and measure the solubility of Aß40. In brief, the method utilizes 96-well filter plates to separate monomeric Aß from aggregated Aß, and the small species are quantified with the amine reactive dye o-phthalaldehyde (OPA). This procedure ensures that solubility is measured for unlabeled species, and makes the assay high-throughput and inexpensive. We demonstrate that the filter plates successfully separate fibrils from monomer, with negligible monomer adsorption, and that OPA can quantify Aß peptides in a concentration range from 40 nM to 20 µM. We also show that adding a methionine residue to the N-terminus of Aß1-40 decreases the solubility by <3-fold. The method will facilitate further solubility studies, and contribute to the understanding of the thermodynamics of amyloid fibril formation.

Proliferation of Tau 304-380 Fragment Aggregates through Autocatalytic Secondary Nucleation.

The self-assembly of the protein tau into neurofibrillary tangles is one of the hallmarks of Alzheimer's disease and related tauopathies. Still, the molecular mechanism of tau aggregation is largely unknown. This problem may be addressed by systematically obtaining reproducible in vitro kinetics measurements under quiescent conditions in the absence of triggering substances. Here, we implement this strategy by developing protocols for obtaining an ultrapure tau fragment (residues 304-380 of tau441) and for performing spontaneous aggregation assays with reproducible kinetics under quiescent conditions. We are thus able to identify the mechanism of fibril formation of the tau 304-380 fragment at physiological pH using fluorescence spectroscopy and mass spectrometry. We find that primary nucleation is slow, and that secondary processes dominate the aggregation process once the initial aggregates are formed. Moreover, our results further show that secondary nucleation of monomers on fibril surfaces dominates over fragmentation of fibrils. Using separate isotopes in monomers and fibrils, through mass spectroscopy measurements, we verify the isotope composition of the intermediate oligomeric species, which reveals that these small aggregates are generated from monomer through secondary nucleation. Our results provide a framework for understanding the processes leading to tau aggregation in disease and for selecting possible tau forms as targets in the development of therapeutic interventions in Alzheimer's disease.