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.

Aß Oligomer Dissociation Is Catalyzed by Fibril Surfaces.

Oligomeric assemblies consisting of only a few protein subunits are key species in the cytotoxicity of neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. Their lifetime in solution and abundance, governed by the balance of their sources and sinks, are thus important determinants of disease. While significant advances have been made in elucidating the processes that govern oligomer production, the mechanisms behind their dissociation are still poorly understood. Here, we use chemical kinetic modeling to determine the fate of oligomers formed in vitro and discuss the implications for their abundance in vivo. We discover that oligomeric species formed predominantly on fibril surfaces, a broad class which includes the bulk of oligomers formed by the key Alzheimer's disease-associated Aß peptides, also dissociate overwhelmingly on fibril surfaces, not in solution as had previously been assumed. We monitor this "secondary nucleation in reverse" by measuring the dissociation of Aß42 oligomers in the presence and absence of fibrils via two distinct experimental methods. Our findings imply that drugs that bind fibril surfaces to inhibit oligomer formation may also inhibit their dissociation, with important implications for rational design of therapeutic strategies for Alzheimer's and other amyloid diseases.

Photo-Induced Cross-Linking of Unmodified α-Synuclein Oligomers.

Photo-induced cross-linking of unmodified proteins (PICUP) has been used in the past to study size distributions of protein assemblies. PICUP may, for example, overcome the significant experimental challenges related to the transient nature, heterogeneity, and low concentration of amyloid protein oligomers relative to monomeric and fibrillar species. In the current study, a reaction chamber was designed, produced, and used for PICUP reaction optimization in terms of reaction conditions and lighting time from ms to s. These efforts make the method more reproducible and accessible and enable the use of shorter reaction times compared to previous studies. We applied the optimized method to an α-synuclein aggregation time course to monitor the relative concentration and size distribution of oligomers over time. The data are compared to the time evolution of the fibril mass concentration, as monitored by thioflavin T fluorescence. At all time points, the smaller the oligomer, the higher its concentration observed after PICUP. Moreover, the total oligomer concentration is highest at short aggregation times, and the decline over time follows the disappearance of monomers. We can therefore conclude that these oligomers form from monomers.

The Bacterial Amyloids Phenol Soluble Modulins from Staphylococcus aureus Catalyze Alpha-Synuclein Aggregation.

Aggregated α-synuclein (α-syn) is the main constituent of Lewy bodies, which are a pathological hallmark of Parkinson's disease (PD). Environmental factors are thought to be potential triggers capable of initiating the aggregation of the otherwise monomeric α-syn. Braak's seminal work redirected attention to the intestine and recent reports of dysbiosis have highlighted the potential causative role of the microbiome in the initiation of pathology of PD. Staphylococcus aureus is a bacterium carried by 30-70% of the general population. It has been shown to produce functional amyloids, called phenol soluble modulins (PSMαs). Here, we studied the kinetics of α-syn aggregation under quiescent conditions in the presence or absence of four different PSMα peptides and observed a remarkable shortening of the lag phase in their presence. Whereas pure α-syn monomer did not aggregate up to 450 h after initiation of the experiment in neither neutral nor mildly acidic buffer, the addition of different PSMα peptides resulted in an almost immediate increase in the Thioflavin T (ThT) fluorescence. Despite similar peptide sequences, the different PSMα peptides displayed distinct effects on the kinetics of α-syn aggregation. Kinetic analyses of the data suggest that all four peptides catalyze α-syn aggregation through heterogeneous primary nucleation. The immunogold electron microscopic analyses showed that the aggregates were fibrillar and composed of α-syn. In addition of the co-aggregated materials to a cell model expressing the A53T α-syn variant fused to GFP was found to catalyze α-syn aggregation and phosphorylation in the cells. Our results provide evidence of a potential trigger of synucleinopathies and could have implications for the prevention of the diseases.

Functional Optimization of Broadly Neutralizing HIV-1 Antibody 10E8 by Promotion of Membrane Interactions.

The 10E8 antibody targets a helical epitope in the membrane-proximal external region (MPER) and transmembrane domain (TMD) of the envelope glycoprotein (Env) subunit gp41 and is among the broadest known neutralizing antibodies against HIV-1. Accordingly, this antibody and its mechanism of action valuably inform the design of effective vaccines and immunotherapies. 10E8 exhibits unusual adaptations to attain specific, high-affinity binding to the MPER at the viral membrane interface. Reversing the charge of the basic paratope surface (from net positive to net negative) reportedly lowered its neutralization potency. Here, we hypothesized that by increasing the net positive charge in similar polar surface patches, the neutralization potency of the antibody may be enhanced. We found that an increased positive charge at this paratope surface strengthened an electrostatic interaction between the antibody and lipid bilayers, enabling 10E8 to interact spontaneously with membranes. Notably, the modified 10E8 antibody did not gain any apparent polyreactivity and neutralized virus with a significantly greater potency. Binding analyses indicated that the optimized 10E8 antibody bound with a higher affinity to the epitope peptide anchored in lipid bilayers and to Env spikes on virions. Overall, our data provide a proof of principle for the rational optimization of 10E8 via manipulation of its interaction with the membrane element of its epitope. However, the observation that a similar mutation strategy did not affect the potency of the first-generation anti-MPER antibody 4E10 shows possible limitations of this principle. Altogether, our results emphasize the crucial role played by the viral membrane in the antigenicity of the MPER-TMD of HIV-1.IMPORTANCE The broadly neutralizing antibody 10E8 blocks infection by nearly all HIV-1 isolates, a capacity which vaccine design seeks to reproduce. Engineered versions of this antibody also represent a promising treatment for HIV infection by passive immunization. Understanding its mechanism of action is therefore important to help in developing effective vaccines and biologics to combat HIV/AIDS. 10E8 engages its helical MPER epitope where the base of the envelope spike submerges into the viral membrane. To enable this interaction, this antibody evolved an unusual property: the ability to interact with the membrane surface. Here, we provide evidence that 10E8 can be made more effective by enhancing its interactions with membranes. Our findings strengthen the idea that to elicit antibodies similar to 10E8, vaccines must reproduce the membrane environment where these antibodies perform their function.