Formation of new amyloid fibrils and oligomers from monomeric protein on the surfaces of existing fibrils is an important driver of many disorders such as Alzheimer's and Parkinson's diseases. The structural basis of this secondary nucleation process, however, is poorly understood. Here, we ask whether secondary nucleation sites are found predominantly at rare growth defects: irregularities in the fibril core structure incorporated during their original assembly. We first demonstrate using the specific inhibitor of secondary nucleation, Brichos, that secondary nucleation sites on Alzheimer's disease-associated fibrils composed of Aβ40 and Aβ42 peptides are rare compared to the number of protein molecules they contain. We then grow Aβ40 fibrils under conditions designed to eliminate most growth defects while leaving the regular fibril morphology unchanged, and confirm the latter using cryo-electron microscopy. We measure both the ability of these annealed fibrils to promote secondary nucleation and the stoichiometry of their secondary nucleation sites, finding that both are greatly reduced as predicted. Re-analysis of published data for other proteins suggests that fibril growth defects may also drive secondary nucleation generally across most amyloids. These findings could unlock structure-based drug design of therapeutics that aim to halt amyloid disorders by inhibiting secondary nucleation sites.

Amyloids are elongated supramolecular protein self-assemblies. Their formation is a non-covalent assembly process and as such is fully reversible. Amyloid formation is associated with several neurodegenerative diseases, and the reversibility is key to maintaining the healthy state. Reversibility is also key to the performance of fibril-based biomaterials and functional amyloids. The reversibility can be observed by a range of spectroscopic, calorimetric, or surface-based techniques using as a starting state either a supersaturated monomer solution or diluted fibrils. Amyloid formation has the characteristics of a phase transition, and we provide some basic formalism for the reversibility and the derivation of the solubility/critical concentration. We also discuss conditions under which the dissociation of amyloids may be so slow that the process can be viewed as practically irreversible, for example, because it is slow relative to the experimental time frame or because the system at hand contains a source for constant monomer addition.

A major hallmark of Alzheimer's disease is the accumulation of aggregated amyloid beta peptide (A beta) in the brain. Here we develop a solubility assay for proteins and measure the solubility of A beta 40. In brief, the method utilizes 96-well filter plates to separate monomeric A beta from aggregated A beta, 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 A beta peptides in a concentration range from 40 nM to 20 mu M. We also show that adding a methionine residue to the N-terminus of A beta 1-40 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.

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 β 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.

An increased understanding of how chaperones affect amyloid aggregation and solubility may yield a new class of therapeutics with the potential to mitigate and treat amyloid diseases. The present study investigates whether DNAJB6 affects the structure of tau304-380C322S fibrils, the solubility of the monomers, and whether co-aggregates of DNAJB6 and tau304-380C322S are formed. A methodology for time-dependent studies of samples towards an apparent equilibrium, separation of monomers and fibrils, and subsequent quantification of monomers through scintillation counting and fluorescent-based detection has been developed.

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 β 42 peptide (Aβ42) 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.

Do the extrinsic factors in cerebral spinal fluid (CSF) from patients suffering from Alzheimer's disease (AD) affect the solubility of tau differently than that of healthy individuals? A method utilizing 3H-labeled tau to quantify the monomer concentration after separation from amyloid fibrils at equilibrium has been established. This is a functional platform on which the effects of extrinsic factors on solubility can be investigated. This method can address questions like how the solubility of tau is affected by the components present in CSF and could elucidate important fundamental aspects of amyloid formation. Determining how much of something you can dissolve in solution can be straightforward: simply keep on adding more of your solute until it starts to precipitate. Proteins on the other hand can often be dissolved above their thermodynamic equilibrium and remain metastable for long periods of time. One way to overcome these energy barriers is to start with high concentration supersaturated solutions of monomers, allow the system enough time to nucleate, elongate and reach equilibrium. Then quantify the amount of monomer left in the solution after separation from the insoluble fibrils. This approach has been successfully adopted in the lab for the Aβ40 peptide. Full-length isoforms of tau are notoriously soluble and require negatively charged inducers to aggregate in vitro. We have overcome the need of inducers by using only the amyloidogenic core of tau, found in ex vivo tangles from patients suffering from AD with Cryo-EM. We have shown that this fragment, spanning amino acids 304-380C322S, aggregates reproducibly through a secondary nucleation dependent manner without the need of inducers. With this model of tau, we aim to investigate the effect of extrinsic factors on solubility.

The transglycosylation behavior and capacity of two clan GH-D α-galactosidases, BoGal36A from the gut bacterium Bacteroides ovatus and Aga27A from the guar plant, was investigated and compared. The enzymes were screened for the ability to use para-nitrophenyl-α-galactoside (pNP-Gal), raffinose and locust bean gum (LBG) galactomannan as glycosyl donors with the glycosyl acceptors methanol, propanol, allyl alcohol, propargyl alcohol and glycerol using mass spectrometry. Aga27A was, in general, more stable in the presence of the acceptors. HPLC analysis was developed and used as a second screening method for reactions using raffinose or LBG as a donor substrate with methanol, propanol and glycerol as acceptors. Time-resolved reactions were set up with raffinose and methanol as the donor and acceptor, respectively, in order to develop an insight into the basic transglycosylation properties, including the ratio between the rate of transglycosylation (methyl galactoside synthesis) and rate of hydrolysis. BoGal36A had a somewhat higher ratio (0.99 compared to 0.71 for Aga27A) at early time points but was indicated to be more prone to secondary (product) hydrolysis in prolonged incubations. The methyl galactoside yield was higher when using raffinose (48% for BoGal36A and 38% for Aga27A) compared to LBG (27% for BoGal36A and 30% for Aga27A).

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 kinetic measurements under quiescent conditions in the absence of triggering substances. Here, we implement this strategy by developing protocols for obtaining an ultra-pure tau fragment (residues 304-380 of tau441, tau AD fragment) 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 AD 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 dominate 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.

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.

The small protein α-synuclein is strongly associated with Parkinson’s disease. This protein is found in inclusion bodies, named lewy bodies inside neurons of people suffering from the disease. α-synuclein is very abundant in the human brain, and its normal function is still elusive and unclear. The factors that trigger the accumulation and fibrillation of this protein into pathogenic inclusion bodies is of great interest in the quest of finding a cure for the disease. In this study the isothermal fibrillation of this protein from monomeric form (found in the brain of healthy people) into aggregated structures (found in people with the disease) was investigated using calorimetry. This yielded insights about the underlying thermodynamics that govern the onset and progression of the disease. This work shows that the process of fibrillation is endothermic (needs energy to proceed) under the investigated conditions and that calorimetry is a method that may be employed in the study of this protein. Attempts were made to investigate whether this protein had an effect on proton permeability across the membrane of small unilamellar vesicles, with a pH gradient between the interior and exterior of the vesicles. In doing so groundwork was laid for developing a vesicle leakage assay using the lytic peptide melittin and studying leakage through isothermal titration calorimetry.