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.

α-Synuclein Oligomers Displace Monomeric α-Synuclein from Lipid Membranes.

Parkinson's disease (PD) is an increasingly prevalent and currently incurable neurodegenerative disorder linked to the accumulation of α-synuclein (αS) protein aggregates in the nervous system. While αS binding to membranes in its monomeric state is correlated to its physiological role, αS oligomerization and subsequent aberrant interactions with lipid bilayers have emerged as key steps in PD-associated neurotoxicity. However, little is known of the mechanisms that govern the interactions of oligomeric αS (OαS) with lipid membranes and the factors that modulate such interactions. This is in large part due to experimental challenges underlying studies of OαS-membrane interactions due to their dynamic and transient nature. Here, we address this challenge by using a suite of microfluidics-based assays that enable in-solution quantification of OαS-membrane interactions. We find that OαS bind more strongly to highly curved, rather than flat, lipid membranes. By comparing the membrane-binding properties of OαS and monomeric αS (MαS), we further demonstrate that OαS bind to membranes with up to 150-fold higher affinity than their monomeric counterparts. Moreover, OαS compete with and displace bound MαS from the membrane surface, suggesting that disruption to the functional binding of MαS to membranes may provide an additional toxicity mechanism in PD. These findings present a binding mechanism of oligomers to model membranes, which can potentially be targeted to inhibit the progression of PD.

Single-molecule digital sizing of proteins in solution.

The physical characterization of proteins in terms of their sizes, interactions, and assembly states is key to understanding their biological function and dysfunction. However, this has remained a difficult task because proteins are often highly polydisperse and present as multicomponent mixtures. Here, we address this challenge by introducing single-molecule microfluidic diffusional sizing (smMDS). This approach measures the hydrodynamic radius of single proteins and protein assemblies in microchannels using single-molecule fluorescence detection. smMDS allows for ultrasensitive sizing of proteins down to femtomolar concentrations and enables affinity profiling of protein interactions at the single-molecule level. We show that smMDS is effective in resolving the assembly states of protein oligomers and in characterizing the size of protein species within complex mixtures, including fibrillar protein aggregates and nanoscale condensate clusters. Overall, smMDS is a highly sensitive method for the analysis of proteins in solution, with wide-ranging applications in drug discovery, diagnostics, and nanobiotechnology.

The seeding barrier between human and Syrian hamster prion protein amyloid fibrils is determined by ß2-α2 loop sequence elements.

Transmissive spongiform encephalopathies (TSE) are a group of neurodegenerative diseases caused by infectious protein particles, known as prions. Prions are formed from cellular prion proteins (PrP) and can be transmitted between different mammalian species. Subsequently, the host's PrPs are then converted to prions, followed by the onset of TSE. Interspecies prion infectivity is governed by the amino acid sequence differences of PrPs and prions' inability to replicate in a host is termed a species barrier. Here, we investigated the amino acid sequence determinants of species barrier between recombinant human (rHuPrP) and hamster (rShaPrP) prion protein amyloid fibrils. We discovered that a unidirectional species barrier between rShaPrP and rHuPrP amyloid fibrils exists. This barrier stems from the difference of amino acid sequences in the conserved ß2-α2 loop region. Our results revealed that individual amino acids in the ß2-α2 loop region are critical for overcoming the barrier between human and hamster prion protein amyloid fibrils in vitro. Furthermore, the barrier was only possible to observe through aggregation kinetics, as the secondary structure rHuPrP fibrils was not affected by the cross-seeding. Overall, we demonstrated the mechanistic pathway behind this interspecies barrier phenomenon, which increases our understanding of prion-related disease development.

A Kinetic Map of the Influence of Biomimetic Lipid Model Membranes on Aß42 Aggregation.

The aggregation of the amyloid ß (Aß) peptide is one of the molecular hallmarks of Alzheimer's disease (AD). Although Aß deposits have mostly been observed extracellularly, various studies have also reported the presence of intracellular Aß assemblies. Because these intracellular Aß aggregates might play a role in the onset and progression of AD, it is important to investigate their possible origins at different locations of the cell along the secretory pathway of the amyloid precursor protein, from which Aß is derived by proteolytic cleavage. Senile plaques found in AD are largely composed of the 42-residue form of Aß (Aß42). Intracellularly, Aß42 is produced in the endoplasmatic reticulum (ER) and Golgi apparatus. Since lipid bilayers have been shown to promote the aggregation of Aß, in this study, we measure the effects of the lipid membrane composition on the in vitro aggregation kinetics of Aß42. By using large unilamellar vesicles to model cellular membranes at different locations, including the inner and outer leaflets of the plasma membrane, late endosomes, the ER, and the Golgi apparatus, we show that Aß42 aggregation is inhibited by the ER and Golgi model membranes. These results provide a preliminary map of the possible effects of the membrane composition in different cellular locations on Aß aggregation and suggest the presence of an evolutionary optimization of the lipid composition to prevent the intracellular aggregation of Aß.