Structural characterization of E22G Aß1-42 fibrils via1H detected MAS NMR.

Amyloid fibrils have been implicated in the pathogenesis of several neurodegenerative diseases, the most prevalent example being Alzheimer's disease (AD). Despite the prevalence of AD, relatively little is known about the structure of the associated amyloid fibrils. This has motivated our studies of fibril structures, extended here to the familial Arctic mutant of Aß1-42, E22G-Aß1-42. We found E22G-AßM0,1-42 is toxic to Escherichia coli, thus we expressed E22G-Aß1-42 fused to the self-cleavable tag NPro in the form of its EDDIE mutant. Since the high surface activity of E22G-Aß1-42 makes it difficult to obtain more than sparse quantities of fibrils, we employed 1H detected magic angle spinning (MAS) nuclear magnetic resonance (NMR) experiments to characterize the protein. The 1H detected 13C-13C methods were first validated by application to fully protonated amyloidogenic nanocrystals of GNNQQNY, and then applied to fibrils of the Arctic mutant of Aß, E22G-Aß1-42. The MAS NMR spectra indicate that the biosynthetic samples of E22G-Aß1-42 fibrils comprise a single conformation with 13C chemical shifts extracted from hCH, hNH, and hCCH spectra that are very similar to those of wild type Aß1-42 fibrils. These results suggest that E22G-Aß1-42 fibrils have a structure similar to that of wild type Aß1-42.

Molecular mechanism of α-synuclein aggregation on lipid membranes revealed.

The central hallmark of Parkinson's disease pathology is the aggregation of the α-synuclein protein, which, in its healthy form, is associated with lipid membranes. Purified monomeric α-synuclein is relatively stable in vitro, but its aggregation can be triggered by the presence of lipid vesicles. Despite this central importance of lipids in the context of α-synuclein aggregation, their detailed mechanistic role in this process has not been established to date. Here, we use chemical kinetics to develop a mechanistic model that is able to globally describe the aggregation behaviour of α-synuclein in the presence of DMPS lipid vesicles, across a range of lipid and protein concentrations. Through the application of our kinetic model to experimental data, we find that the reaction is a co-aggregation process involving both protein and lipids and that lipids promote aggregation as much by enabling fibril elongation as by enabling their initial formation. Moreover, we find that the primary nucleation of lipid-protein co-aggregates takes place not on the surface of lipid vesicles in bulk solution but at the air-water and/or plate interfaces, where lipids and proteins are likely adsorbed. Our model forms the basis for mechanistic insights, also in other lipid-protein co-aggregation systems, which will be crucial in the rational design of drugs that inhibit aggregate formation and act at the key points in the α-synuclein aggregation cascade.

Interplay of α-synuclein with Lipid Membranes: Cooperative Adsorption, Membrane Remodeling and Coaggregation.

α-Synuclein is a small neuronal protein enriched at presynaptic termini. It is hypothesized to play a role in neurotransmitter release and synaptic vesicle cycling, while the formation of α-synuclein amyloid fibrils is associated with several neurodegenerative diseases, most notably Parkinson's Disease. The molecular mechanisms of both the physiological and pathological functions of α-synuclein remain to be fully understood, but in both cases, interactions with membranes play an important role. In this Perspective, we discuss several aspects of α-synuclein interactions with lipid membranes including cooperative adsorption, membrane remodeling and α-synuclein amyloid fibril formation in the presence of lipid membranes. We highlight the coupling between the different phenomena and their interplay in the context of physiological and pathological functions of α-synuclein.

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.

Structural characterisation of α-synuclein-membrane interactions and the resulting aggregation using small angle scattering.

The presence of amyloid fibrils is a hallmark of several neurodegenerative diseases. Some amyloidogenic proteins, such as α-synuclein and amyloid ß, interact with lipids, and this interaction can strongly favour the formation of amyloid fibrils. In particular the primary nucleation step, i.e. the de novo formation of amyloid fibrils, has been shown to be accelerated by lipids. However, the exact mechanism of this acceleration is still mostly unclear. Here we use a range of scattering methods, such as dynamic light scattering (DLS) and small angle X-ray and neutron scattering (SAXS and SANS) to obtain structural information on the binding of α-synuclein to model membranes formed from negatively charged lipids and their co-assembly into amyloid fibrils. We find that the model membranes take an active role in the reaction. The binding of α synuclein to the model membranes immediately induces a major structural change in the lipid assembly, which leads to a break-up into small and mostly disc- or rod-like lipid-protein particles. This transition can be reversed by temperature changes or proteolytic protein removal. Incubation of the small lipid-α-synuclein particles for several hours, however, leads to amyloid fibril formation, whereby the lipids are incorporated into the amyloid fibrils.

Self-replication of Aß42 aggregates occurs on small and isolated fibril sites.

Self-replication of amyloid fibrils via secondary nucleation is an intriguing physicochemical phenomenon in which existing fibrils catalyze the formation of their own copies. The molecular events behind this fibril surface-mediated process remain largely inaccessible to current structural and imaging techniques. Using statistical mechanics, computer modeling, and chemical kinetics, we show that the catalytic structure of the fibril surface can be inferred from the aggregation behavior in the presence and absence of a fibril-binding inhibitor. We apply our approach to the case of Alzheimer's A[Formula: see text] amyloid fibrils formed in the presence of proSP-C Brichos inhibitors. We find that self-replication of A[Formula: see text] fibrils occurs on small catalytic sites on the fibril surface, which are far apart from each other, and each of which can be covered by a single Brichos inhibitor.

A Relationship between the Structures and Neurotoxic Effects of Aß Oligomers Stabilized by Different Metal Ions.

Oligomeric assemblies of the amyloid ß peptide (Aß) have been investigated for over two decades as possible neurotoxic agents in Alzheimer's disease. However, due to their heterogeneous and transient nature, it is not yet fully established which of the structural features of these oligomers may generate cellular damage. Here, we study distinct oligomer species formed by Aß40 (the 40-residue form of Aß) in the presence of four different metal ions (Al3+, Cu2+, Fe2+, and Zn2+) and show that they differ in their structure and toxicity in human neuroblastoma cells. We then describe a correlation between the size of the oligomers and their neurotoxic activity, which provides a type of structure-toxicity relationship for these Aß40 oligomer species. These results provide insight into the possible role of metal ions in Alzheimer's disease by the stabilization of Aß oligomers.

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.

The role of phosphorylation in calmodulin-mediated gating of human AQP0.

Aquaporin-0 (AQP0) is the main water channel in the mammalian lens and is involved in accommodation and maintaining lens transparency. AQP0 binds the Ca2+-sensing protein calmodulin (CaM) and this interaction is believed to gate its water permeability by closing the water-conducting pore. Here, we express recombinant and functional human AQP0 in Pichia pastoris and investigate how phosphorylation affects the interaction with CaM in vitro as well as the CaM-dependent water permeability of AQP0 in proteoliposomes. Using microscale thermophoresis and surface plasmon resonance technology we show that the introduction of the single phospho-mimicking mutations S229D and S235D in AQP0 reduces CaM binding. In contrast, CaM interacts with S231D with similar affinity as wild type, but in a different manner. Permeability studies of wild-type AQP0 showed that the water conductance was significantly reduced by CaM in a Ca2+-dependent manner, whereas AQP0 S229D, S231D and S235D were all locked in an open state, insensitive to CaM. We propose a model in which phosphorylation of AQP0 control CaM-mediated gating in two different ways (1) phosphorylation of S229 or S235 abolishes binding (the pore remains open) and (2) phosphorylation of S231 results in CaM binding without causing pore closure, the functional role of which remains to be elucidated. Our results suggest that site-dependent phosphorylation of AQP0 dynamically controls its CaM-mediated gating. Since the level of phosphorylation increases towards the lens inner cortex, AQP0 may become insensitive to CaM-dependent gating along this axis.

The density of anionic lipids modulates the adsorption of α-Synuclein onto lipid membranes.

α-Synuclein is an intrinsically disordered presynaptic protein associated with Parkinson's disease. The physiological role of α-Synuclein is not fully understood, but the protein is known to interact with lipid membranes. We here study how membrane charge affects the adsorption of α-Synuclein to (i) supported lipid bilayers and (ii) small unilamellar vesicles with varying amounts of anionic lipids. The results showed that α-Synuclein adsorbs onto membranes containing ≥5% anionic phosphatidylserine (DOPS) lipids, but not to membranes containing ≤1% DOPS. The density of adsorbed α-Synuclein increased steadily with the DOPS content up to 20% DOPS, after which it leveled off. The vesicles were saturated with α-Synuclein at a 3-5 times higher protein density compared to the supported bilayers, which suggests that a more deformable membrane binds more α-Synuclein. Altogether, the results show that both membrane charge density and flexibility influence the association of α-Synuclein to lipid membranes.

Ganglioside Micelles Affect Amyloid ß Aggregation by Coassembly.

Amyloid ß peptide (Aß) is the crucial protein component of extracellular plaques in Alzheimer's disease. The plaques also contain gangliosides lipids, which are abundant in membranes of neuronal cells and in cell-derived vesicles and exosomes. When present at concentrations above its critical micelle concentration (cmc), gangliosides can occur as mixed micelles. Here, we study the coassembly of the ganglioside GM1 and the Aß peptides Aß40 and 42 by means of microfluidic diffusional sizing, confocal microscopy, and cryogenic transmission electron microscopy. We also study the effects of lipid-peptide interactions on the amyloid aggregation process by fluorescence spectroscopy. Our results reveal coassembly of GM1 lipids with both Aß monomers and Aß fibrils. The results of the nonseeded kinetics experiments show that Aß40 aggregation is delayed with increasing GM1 concentration, while that of Aß42 is accelerated. In seeded aggregation reactions, the addition of GM1 leads to a retardation of the aggregation process of both peptides. Thus, while the effect on nucleation differs between the two peptides, GM1 may inhibit the elongation of both types of fibrils. These results shed light on glycolipid-peptide interactions that may play an important role in Alzheimer's pathology.

The C-terminal domain of the antiamyloid chaperone DNAJB6 binds to amyloid-ß peptide fibrils and inhibits secondary nucleation.

The DNAJB6 chaperone inhibits fibril formation of aggregation-prone client peptides through interaction with aggregated and oligomeric forms of the amyloid peptides. Here, we studied the role of its C-terminal domain (CTD) using constructs comprising either the entire CTD or the first two or all four of the CTD ß-strands grafted onto a scaffold protein. Each construct was expressed as WT and as a variant with alanines replacing five highly conserved and functionally important serine and threonine residues in the first ß-strand. We investigated the stability, oligomerization, antiamyloid activity, and affinity for amyloid-ß (Aß42) species using optical spectroscopy, native mass spectrometry, chemical crosslinking, and surface plasmon resonance technology. While DNAJB6 forms large and polydisperse oligomers, CTD was found to form only monomers, dimers, and tetramers of low affinity. Kinetic analyses showed a shift in inhibition mechanism. Whereas full-length DNAJB6 activity is dependent on the serine and threonine residues and efficiently inhibits primary and secondary nucleation, all CTD constructs inhibit secondary nucleation only, independently of the serine and threonine residues, although their dimerization and thermal stabilities are reduced by alanine substitution. While the full-length DNAJB6 inhibition of primary nucleation is related to its propensity to form coaggregates with Aß, the CTD constructs instead bind to Aß42 fibrils, which affects the nucleation events at the fibril surface. The retardation of secondary nucleation by DNAJB6 can thus be ascribed to the first two ß-strands of its CTD, whereas the inhibition of primary nucleation is dependent on the entire protein or regions outside the CTD.

Retardation of Aß42 fibril formation by apolipoprotein A-I and recombinant HDL particles.

The double nucleation mechanism of amyloid ß (Aß) peptide aggregation is retained from buffer to cerebrospinal fluid (CSF) but with reduced rate of all microscopic processes. Here, we used a bottom-up approach to identify retarding factors in CSF. We investigated the Aß42 fibril formation as a function of time in the absence and presence of apolipoprotein A-I (ApoA-I), recombinant high-density lipoprotein (rHDL) particles, or lipid vesicles. A retardation was observed in the presence of ApoA-I or rHDL particles, most pronounced with ApoA-I, but not with lipid vesicles. Global kinetic analysis implies that rHDL interferes with secondary nucleation. The effect of ApoA-I could best be described as an interference with secondary and to a smaller extent primary nucleation. Using surface plasmon resonance and microfluidics diffusional sizing analyses, we find that both rHDL and ApoA-I interact with Aß42 fibrils but not Aß42 monomer, thus the effect on kinetics seems to involve interference with the catalytic surface for secondary nucleation. The Aß42 fibrils were imaged using cryogenic-electron microscopy and found to be longer when formed in the presence of ApoA-I or rHDL, compared to formation in buffer. A retarding effect, as observed in CSF, could be replicated using a simpler system, from key components present in CSF but purified from a CSF-free host. However, the effect of CSF is stronger implying the presence of additional retarding factors.

On the micelle formation of DNAJB6b.

The human chaperone DNAJB6b increases the solubility of proteins involved in protein aggregation diseases and suppresses the nucleation of amyloid structures. Due to such favourable properties, DNAJB6b has gained increasing attention over the past decade. The understanding of how DNAJB6b operates on a molecular level may aid the design of inhibitors against amyloid formation. In this work, fundamental aspects of DNAJB6b self-assembly have been examined, providing a basis for future experimental designs and conclusions. The results imply the formation of large chaperone clusters in a concentration-dependent manner. Microfluidic diffusional sizing (MDS) was used to evaluate how DNAJB6b average hydrodynamic radius varies with concentration. We found that, in 20 mM sodium phosphate buffer, 0.2 mM EDTA, at pH 8.0 and room temperature, DNAJB6b displays a micellar behaviour, with a critical micelle concentration (CMC) of around 120 nM. The average hydrodynamic radius appears to be concentration independent between ∼10 µM and 100 µM, with a mean radius of about 12 nm. The CMC found by MDS is supported by native agarose gel electrophoresis and the size distribution appears bimodal in the DNAJB6b concentration range ∼100 nM to 4 µM.

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.

Direct observation of secondary nucleation along the fibril surface of the amyloid ß 42 peptide.

Alzheimer's disease is a neurodegenerative condition which involves heavy neuronal cell death linked to oligomers formed during the aggregation process of the amyloid ß peptide 42 (Aß42). The aggregation of Aß42 involves both primary and secondary nucleation. Secondary nucleation dominates the generation of oligomers and involves the formation of new aggregates from monomers on catalytic fibril surfaces. Understanding the molecular mechanism of secondary nucleation may be crucial in developing a targeted cure. Here, the self-seeded aggregation of WT Aß42 is studied using direct stochastic optical reconstruction microscopy (dSTORM) with separate fluorophores in seed fibrils and monomers. Seeded aggregation proceeds faster than nonseeded reactions because the fibrils act as catalysts. The dSTORM experiments show that monomers grow into relatively large aggregates on fibril surfaces along the length of fibrils before detaching, thus providing a direct observation of secondary nucleation and growth along the sides of fibrils. The experiments were repeated for cross-seeded reactions of the WT Aß42 monomer with mutant Aß42 fibrils that do not catalyze the nucleation of WT monomers. While the monomers are observed by dSTORM to interact with noncognate fibril surfaces, we fail to notice any growth along such fibril surfaces. This implies that the failure to nucleate on the cognate seeds is not a lack of monomer association but more likely a lack of structural conversion. Our findings support a templating role for secondary nucleation, which can only take place if the monomers can copy the underlying parent structure without steric clashes or other repulsive interactions between nucleating monomers.

Amyloids and protein aggregation.

A general discovery in protein science in the past few decades has been the finding that a number of unrelated proteins and peptides all have a marked propensity to form amyloid fibrils in vivo and in vitro. These structures have become known as the pathological hallmark of some of the most prevalent neurodegenerative diseases. More recently, the process of amyloid formation has been demystified through a number of key mechanistic findings, some of which are highlighted in this themed collection.

Morphology-Dependent Interactions between α-Synuclein Monomers and Fibrils.

Amyloid fibrils may adopt different morphologies depending on the solution conditions and the protein sequence. Here, we show that two chemically identical but morphologically distinct α-synuclein fibrils can form under identical conditions. This was observed by nuclear magnetic resonance (NMR), circular dichroism (CD), and fluorescence spectroscopy, as well as by cryo-transmission electron microscopy (cryo-TEM). The results show different surface properties of the two morphologies, A and B. NMR measurements show that monomers interact differently with the different fibril surfaces. Only a small part of the N-terminus of the monomer interacts with the fibril surface of morphology A, compared to a larger part of the monomer for morphology B. Differences in ThT binding seen by fluorescence titrations, and mesoscopic structures seen by cryo-TEM, support the conclusion of the two morphologies having different surface properties. Fibrils of morphology B were found to have lower solubility than A. This indicates that fibrils of morphology B are thermodynamically more stable, implying a chemical potential of fibrils of morphology B that is lower than that of morphology A. Consequently, at prolonged incubation time, fibrils of morphology B remained B, while an initially monomorphic sample of morphology A gradually transformed to B.

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.