α-synuclein (αS) is an intrinsically disordered protein whose fibrillar aggregates are the major constituents of Lewy bodies in Parkinson's disease. Although the specific function of αS is still ...unclear, a general consensus is forming that it has a key role in regulating the process of neurotransmitter release, which is associated with the mediation of synaptic vesicle interactions and assembly. Here we report the analysis of wild-type αS and two mutational variants linked to familial Parkinson's disease to describe the structural basis of a molecular mechanism enabling αS to induce the clustering of synaptic vesicles. We provide support for this 'double-anchor' mechanism by rationally designing and experimentally testing a further mutational variant of αS engineered to promote stronger interactions between synaptic vesicles. Our results characterize the nature of the active conformations of αS that mediate the clustering of synaptic vesicles, and indicate their relevance in both functional and pathological contexts.
This review summarises our current understanding of the underlying and universal mechanism by which newly synthesised proteins achieve their biologically functional states. Protein molecules, ...however, all have a finite tendency either to misfold, or to fail to maintain their correctly folded states, under some circumstances. This article describes some of the consequences of such behaviour, particularly in the context of the aggregation events that are frequently associated with aberrant folding. It focuses in particular on the emerging links between protein aggregation and the increasingly prevalent forms of debilitating disease with which it is now known to be associated.
'In dopaminergic neurons, α-synuclein (αS) partitions between a disordered cytosolic state and a lipid-bound state. Binding of αS to membrane phospholipids is implicated in its functional role in ...synaptic regulation, but also impacts fibril formation associated with Parkinson's disease. We describe here a solution NMR study in which αS is added to small unilamellar vesicles of a composition mimicking synaptic vesicles; the results provide evidence for multiple distinct phospholipid-binding modes of αS. Exchange between the free state and the lipid-bound αS state, and between different bound states is slow on the NMR timescale, being in the range of 1–10 s
−
1
. Partitioning of the binding modes is dependent on lipid/αS stoichiometry, and tight binding with slow-exchange kinetics is observed at stoichiometries as low as 2:1. In all lipid-bound states, a segment of residues starting at the N-terminus of αS adopts an α-helical conformation, while succeeding residues retain the characteristics of a random coil. The 40 C-terminal residues remain dynamically disordered, even at high-lipid concentrations, but can also bind to lipids to an extent that appears to be determined by the fraction of
cis X-Pro peptide bonds in this region. While lipid-bound αS exhibits dynamic properties that preclude its direct observation by NMR, its exchange with the NMR-visible free form allows for its indirect characterization. Rapid amide–amide nuclear Overhauser enhancement buildup points to a large α-helical conformation, and a distinct increase in fluorescence anisotropy attributed to Tyr39 indicates an ordered environment for this “dark state.” Titration of αS with increasing amounts of lipids suggests that the binding mode under high-lipid conditions remains qualitatively similar to that in the low-lipid case. The NMR data appear incompatible with the commonly assumed model where αS lies in an α-helical conformation on the membrane surface and instead suggest that considerable remodeling of the vesicles is induced by αS.
Significance Certain oligomeric species generated during the self-assembly of specific proteins into ordered fibrillar aggregates are likely to be key players in the initiation and spreading of ...neurodegenerative diseases. We have purified stable toxic oligomeric species of α-synuclein and defined and minimized their degree of heterogeneity, which has allowed us to identify distinct subgroups of oligomers and determine their structural properties and three-dimensional molecular architectures. All the oligomeric subgroups possess approximately cylindrical architectures with marked similarities to amyloid fibrils, suggesting that these types of oligomers are kinetically trapped during protein self-assembly. The relative stabilities and inherent pathological roles of different amyloid oligomers are likely to result from the multiplicity of pathways of the misfolding process and the remarkably slow rates of structural conversions.
We describe the isolation and detailed structural characterization of stable toxic oligomers of α-synuclein that have accumulated during the process of amyloid formation. Our approach has allowed us to identify distinct subgroups of oligomers and to probe their molecular architectures by using cryo-electron microscopy (cryoEM) image reconstruction techniques. Although the oligomers exist in a range of sizes, with different extents and nature of β-sheet content and exposed hydrophobicity, they all possess a hollow cylindrical architecture with similarities to certain types of amyloid fibril, suggesting that the accumulation of at least some forms of amyloid oligomers is likely to be a consequence of very slow rates of rearrangement of their β-sheet structures. Our findings reveal the inherent multiplicity of the process of protein misfolding and the key role the β-sheet geometry acquired in the early stages of the self-assembly process plays in dictating the kinetic stability and the pathological nature of individual oligomeric species.
The aggregation of the 42-residue form of the amyloid-β peptide (Aβ42) is a pivotal event in Alzheimer’s disease (AD). The use of chemical kinetics has recently enabled highly accurate ...quantifications of the effects of small molecules on specific microscopic steps in Aβ42 aggregation. Here, we exploit this approach to develop a rational drug discovery strategy against Aβ42 aggregation that uses as a readout the changes in the nucleation and elongation rate constants caused by candidate small molecules. We thus identify a pool of compounds that target specific microscopic steps in Aβ42 aggregation. We then test further these small molecules in human cerebrospinal fluid and in a Caenorhabditis elegans model of AD. Our results show that this strategy represents a powerful approach to identify systematically small molecule lead compounds, thus offering an appealing opportunity to reduce the attrition problem in drug discovery.
The conversion of peptides or proteins from their soluble native states into intractable amyloid deposits is associated with a wide range of human disorders. Misfolded protein oligomers formed during ...the process of aggregation have been identified as the primary pathogenic agents in many such conditions. Here, we show the existence of a quantitative relationship between the degree of binding to neuronal cells of different types of oligomers formed from a model protein, HypF-N, and the GM1 content of the plasma membranes. In addition, remarkably similar behavior is observed for oligomers of the Aβ42 peptide associated with Alzheimer's disease. Further analysis has revealed the existence of a linear correlation between the level of the influx of Ca(2+) across neuronal membranes that triggers cellular damage, and the fraction of oligomeric species bound to the membrane. Our findings indicate that the susceptibility of neuronal cells to different types of misfolded oligomeric assemblies is directly related to the extent of binding of such oligomers to the cellular membrane.
The hydrophobic effect is a major driving force in protein folding. A complete understanding of this effect requires the description of the conformational states of water and protein molecules at ...different temperatures. Towards this goal, we characterise the cold and hot denatured states of a protein by modelling NMR chemical shifts using restrained molecular dynamics simulations. A detailed analysis of the resulting structures reveals that water molecules in the bulk and at the protein interface form on average the same number of hydrogen bonds. Thus, even if proteins are 'large' particles (in terms of the hydrophobic effect, i.e. larger than 1 nm), because of the presence of complex surface patterns of polar and non-polar residues their behaviour can be compared to that of 'small' particles (i.e. smaller than 1 nm). We thus find that the hot denatured state is more compact and richer in secondary structure than the cold denatured state, since water at lower temperatures can form more hydrogen bonds than at high temperatures. Then, using Φ-value analysis we show that the structural differences between the hot and cold denatured states result in two alternative folding mechanisms. These findings thus illustrate how the analysis of water-protein hydrogen bonds can reveal the molecular origins of protein behaviours associated with the hydrophobic effect.
The misfolding and aberrant assembly of peptides and proteins into fibrillar aggregates is the hallmark of many pathologies. Fibril formation is accompanied by oligomeric species thought to be the ...primary pathogenic agents in many of these diseases. With the aim of identifying the structural determinants responsible for the toxicity of misfolded oligomers, we created 12 oligomeric variants from the N-terminal domain of the E. coli HypF protein (HypF-N) by replacing one or more charged amino acid residues with neutral apolar residues and allowing the mutated proteins to aggregate under two sets of conditions. The resulting oligomeric species have different degrees of cytotoxicity when added to the extracellular medium of the cells, as assessed by the extent of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, apoptosis, and influx of Ca2+ into the cells. The structural properties of the oligomeric variants were characterized by evaluating their surface hydrophobicity with 8-anilinonaphthalene-1-sulfonate (ANS) binding and by measuring their size by means of turbidimetry as well as light scattering. We find that increases in the surface hydrophobicity of the oligomers following mutation can promote the formation of larger assemblies and that the overall toxicity correlates with a combination of both surface hydrophobicity and size, with the most toxic oligomers having high hydrophobicity and small size. These results have allowed the relationships between these three parameters to be studied simultaneously and quantitatively, and have enabled the generation of an equation that is able to rationalize and even predict toxicity of the oligomers resulting from their surface hydrophobicity and size.
This paper describes research relating to the major recall of pet food that occurred in Spring 2007 in North America. Clinical observations of acute renal failure in cats and dogs were associated ...with consumption of wet pet food produced by a contract manufacturer producing for a large number of companies. The affected lots of food had been formulated with wheat gluten originating from China. Pet food and gluten were analyzed for contaminants using several configurations of high-performance liquid chromatography (HPLC) and mass spectrometry (MS), which revealed a number of simple triazine compounds, principally melamine and cyanuric acid, with lower concentrations of ammeline, ammelide, ureidomelamine, and N-methylmelamine. Melamine and cyanuric acid, have been tested and do not produce acute renal toxicity. Some of the triazines have poor solubility, as does the compound melamine cyanurate. Pathological evaluation of cats and dogs that had died from the acute renal failure indicated the presence of crystals in kidney tubules. We hypothesized that these crystals were composed of the poorly soluble triazines, a melamine-cyanuric acid complex, or a combination. Sprague dawley rats were given up to 100 mg/kg ammeline or ammelide alone, a mixture of melamine and cyanuric acid (400/400 mg/kg/day), or a mixture of all four compounds (400 mg/kg/day melamine, 40 mg/kg/day of the others). Neither ammeline nor ammelide alone produced any renal effects, but the mixtures produced significant renal damage and crystals in nephrons. HPLC-MS/MS confirmed the presence of melamine and cyanuric acid in the kidney. Infrared microspectroscopy on individual crystals from rat or cat (donated material from a veterinary clinic) kidneys confirmed that they were melamine-cyanuric acid cocrystals. Crystals from contaminated gluten produced comparable spectra. These results establish the causal link between the contaminated gluten and the adverse effects and provide a mechanistic explanation for how two apparently innocuous compounds could have adverse effects in combination, that is, by forming an insoluble precipitate in renal tubules leading to progressive tubular blockage and degeneration.
The ability of many proteins to convert from their functional soluble state to amyloid fibrils can be attributed to inter-molecular beta strand formation. Such amyloid formation is associated with ...neurodegenerative disorders like Alzheimer's and Parkinson's. Molecular modelling can play a key role in providing insight into the factors that make proteins prone to fibril formation. However, fully atomistic models are computationally too expensive to capture the length and time scales associated with fibril formation. As the ability to form fibrils is the rule rather than the exception, much insight can be gained from the study of coarse-grained models that capture the key generic features associated with amyloid formation. Here we present a simple lattice model that can capture both protein folding and beta strand formation. Unlike standard lattice models, this model explicitly incorporates the formation of hydrogen bonds and the directionality of side chains. The simplicity of our model makes it computationally feasible to investigate the interplay between folding, amorphous aggregation and fibril formation, and maintains the capability of classic lattice models to simulate protein folding with high specificity. In our model, the folded proteins contain structures that resemble naturally occurring beta-sheets, with alternating polar and hydrophobic amino acids. Moreover, fibrils with intermolecular cross-beta strand conformations can be formed spontaneously out of multiple short hydrophobic peptide sequences. Both the formation of hydrogen bonds in folded structures and in fibrils is strongly dependent on the amino acid sequence, indicating that hydrogen-bonding interactions alone are not strong enough to initiate the formation of beta sheets. This result agrees with experimental observations that beta sheet and amyloid formation is strongly sequence dependent, with hydrophobic sequences being more prone to form such structures. Our model should open the way to a systematic study of the interplay between the factors that lead to amyloid formation.
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