[show abstract][hide abstract] ABSTRACT: Parkinson's Disease (PD) is a neurodegenerative movement disorder affecting millions of people worldwide. One of the key players in the development of the disease is the protein α-synuclein (aSN), which aggregates in the brain of PD patients. The aSN mutant A30P has been reported to cause early-onset familial PD and shows different aggregation behavior compared to wt aSN. Here we use a multidisciplinary approach to compare the aggregation process of wt and A30P aSN. In agreement with previous studies, we observe an initial lag phase followed by a continuous structural development of fibrils until reaching an apparent monomer-aggregate equilibrium state and a plateau in Thioflavin T (ThT) fluorescence intensity. However, at later timepoints A30P shows greater propensity than αSN wt to form dense bundled fibril networks. Combining small angle x-ray scattering, x-ray fibre diffraction and linear dichroism, we demonstrate that while the microscopic structure of the individual fibril essentially remains constant throughout the experiment, the formation of dense A30P fibril networks occur through a continuous assembly pathway while the formation of less dense wt fibril networks with fewer contact points follows a continuous path during the elongation phase and a second rearrangement phase after reaching the ThT fluorescence plateau. Our work thus highlights that structural rearrangements proceed beyond the plateau in ThT-based monitoring of the fibrillation process, and the density and morphology of the resulting fibril networks is highly dependent on the aSN form studied.
[show abstract][hide abstract] ABSTRACT: The 219-residue protein p25α stimulates the fibrillation of α-synuclein (αSN) in vitro and colocalizes with it in several α-synucleinopathies. Although p25α does not fibrillate by itself under native conditions in vitro, αSN-free p25α aggregates have also been observed in vivo in, for example, multiple system atrophy. To investigate which environmental conditions might trigger this aggregation, we investigated the effect of polyanionic biomolecules on p25α aggregation. Heparin, polyglutamate, arachidonic acid micelles, and RNA all induce p25α aggregation. More detailed studies using heparin as template for aggregation reveal that a minimum of 10-14 heparin monosaccharide units per heparin polymer are required. Bona fide fibrils are only formed at intermediate heparin concentrations, possibly because an excess of heparin binding sites blocks the inter-p25α contacts required for amyloid formation. Other polyanions also show an optimum for amyloid formation. Aggregation involves only modest structural changes according to both spectroscopic and proteolytic experiments. The aggregates do not seed aggregation of heparin-free p25α, suggesting that heparin is required in stoichiometric amounts to form organized structures. We are able to reproduce these observations in a model involving two levels of binding of p25α to heparin. We conclude that the modest structural changes that p25α undergoes can promote weak intermolecular contacts and that polyanions such as heparin play a central role in stabilizing these aggregates but in multiple ways, leading to different types of aggregates. This highlights the role of non-protein components in promoting protein aggregation in vivo.
Journal of Molecular Biology 02/2012; 421(4-5):601-15. · 3.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: One of the major hallmarks of Parkinson disease is aggregation of the protein α-synuclein (αSN). Aggregate cytotoxicity has been linked to an oligomeric species formed at early stages in the aggregation process. Here we follow the fibrillation process of αSN in solution over time using small angle X-ray scattering and resolve four major coexisting species in the fibrillation process, namely monomer, dimer, fibril and an oligomer. By ab initio modeling to fit the data, we obtain a low-resolution structure of a symmetrical and slender αSN fibril in solution, consisting of a repeating unit with a maximal distance of 900 Å and a diameter of ∼180 Å. The same approach shows the oligomer to be shaped like a wreath, with a central channel and with dimensions corresponding to the width of the fibril. The structure, accumulation and decay of this oligomer is consistent with an on-pathway role for the oligomer in the fibrillation process. We propose an oligomer-driven αSN fibril formation mechanism, where the fibril is built from the oligomers. The wreath-shaped structure of the oligomer highlights its potential cytotoxicity by simple membrane permeabilization. This is confirmed by the ability of the purified oligomer to disrupt liposomes. Our results provide the first structural description in solution of a potentially cytotoxic oligomer, which accumulates during the fibrillation of αSN.
Proceedings of the National Academy of Sciences 02/2011; 108(8):3246-51. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: This review describes different ways to achieve and monitor reproducible aggregation of α-synuclein, a key protein in the development of Parkinson's disease. For most globular proteins, aggregation is promoted by partially denaturing conditions which compromise the native state without destabilizing the intermolecular contacts required for accumulation of regular amyloid structure. As a natively disordered protein, α-synuclein can fibrillate under physiological conditions and this process is actually stimulated by conditions that promote structure formation, such as low pH, ions, polyamines, anionic surfactants, fluorinated alcohols and agitation. Reproducibility is a critical issue since α-synuclein shows erratic fibrillation behavior on its own. Agitation in combination with glass beads significantly reduces the variability of aggregation time curves, but the most reproducible aggregation is achieved by sub-micellar concentrations of SDS, which promote the rapid formation of small clusters of α-synuclein around shared micelles. Although the fibrils produced this way have a different appearance and secondary structure, they are rich in cross-β structure and are amenable to high-throughput screening assays. Although such assays at best provide a very simplistic recapitulation of physiological conditions, they allow the investigator to focus on well-defined molecular events and may provide the opportunity to identify, e.g. small molecule inhibitors of aggregation that affect these steps. Subsequent experiments in more complex cellular and whole-organism environments can then validate whether there is any relation between these molecular interactions and the broader biological context.
[show abstract][hide abstract] ABSTRACT: We present an analysis of the conformational and aggregative properties of an Aß concatemer (Con-Alz) of interest for vaccine development against Alzheimer's disease. Con-Alz consists of 3 copies of the 43 residues of the Aß peptide separated by the P2 and P30 T-cell epitopes from the tetanus toxin. Even in the presence of high concentrations of denaturants or fluorinated alcohols, Con-Alz has a very high propensity to form aggregates which slowly coalesce over time with changes in secondary, tertiary and quaternary structure. Only micellar concentrations of SDS were able to inhibit aggregation. The increase in the ability to bind the fibril-binding dye ThT increases without lag time, which is characteristic of relatively amorphous aggregates. Confirming this, electron microscopy reveals that Con-Alz adopts a morphology resembling truncated protofibrils after prolonged incubation, but it is unable to assemble into classical amyloid fibrils. Despite its high propensity to aggregate, Con-Alz does not show any significant ability to permeabilize vesicles, which for fibrillating proteins is taken to be a key factor in aggregate cytotoxicity and is attributed to oligomers formed at an early stage in the fibrillation process. Physically linking multiple copies of the Aß-peptide may thus sterically restrict Con-Alz against forming cytotoxic oligomers, forcing it instead to adopt a less well-organized assembly of intermeshed polypeptide chains.
Biochimica et Biophysica Acta 10/2010; 1804(10):2025-35. · 4.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: A structural investigation of the sodium dodecyl sulfate (SDS)-induced fibrillation of alpha-synuclein (alphaSN), a 140-amino-acid protein implicated in Parkinson's disease, has been performed. Spectroscopic analysis has been combined with isothermal titration calorimetry, small-angle X-ray scattering, and transmission electron microscopy to elucidate a fibrillation pathway that is remarkably different from the fibrillation pathway in the absence of SDS. Fibrillation occurs most extensively and most rapidly (starting within 45 min) under conditions where 12 SDS molecules are bound per alphaSN molecule, which is also the range where SDS binding is associated with the highest enthalpy. Fibrillation is only reduced in proportion to the fraction of SDS below 25 mol% SDS in mixed surfactant mixtures with nonionic surfactants and is inhibited by formation of bulk micelles and induction of alpha-helical structure. In this fibrillogenic complex, 4 alphaSN molecules initially associate with 40-50 SDS molecules to form a shared micelle that gradually grows in size. The complex initially exhibits a mixture of random coil and alpha-helix, but incubation results in a structural conversion into beta-sheet structure and concomitant formation of thioflavin-T-binding fibrils over a period of several hours. Based on small-angle X-ray scattering, the aggregates elongate as a beads-on-a-string structure in which individual units of ellipsoidal SDS-alphaSN are bridged by strings of the protein, so that aggregates nucleate around the surface of protein-stabilized micelles. Thus, fibrillation in this case occurs by a process of continuous accretion rather than by the rate-limiting accumulation of a distinct nucleus. The morphology of the SDS-induced fibrils does not exhibit the classical rod-like structures formed by alphaSN when aggregated by agitation in the absence of SDS. The SDS-induced fibrils have a flexible worm-like appearance, which can be converted into classical straight fibrils by continuous agitation. SDS-induced fibrillation represents an alternative and highly reproducible mechanism for fibrillation where protein association is driven by the formation of shared micelles, which subsequently allows the formation of beta-sheet structures that presumably link individual micelles. This illustrates that protein fibrillation may occur by remarkably different mechanisms, testifying to the versatility of this process.
Journal of Molecular Biology 08/2010; 401(1):115-33. · 3.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: There is great interest in developing reproducible high-throughput screens to identify small molecular inhibitors of protein fibrillization and aggregation for possible therapy against deposition diseases such as Alzheimer's and Parkinson's (PD). We have made a methodical analysis of factors increasing the reproducibility of the fibrillization of alpha-synuclein (alphaSN), a 140-amino-acid protein implicated in PD and notorious for its erratic fibrillization behavior. Salts and polyanionic polymers do not significantly improve the quality of the assay. However, an orbital agitation mode in the plate reader is a crucial first step toward reproducible alphaSN fibrillization. Higher reproducibility is achieved by the addition of glass beads, as evaluated by the percentage standard deviation of the nucleation and elongation rate constants and the end-stage fluorescence intensity of the fibril-binding dye thioflavin T (ThT). The highest reproducibility is obtained by either seeding the solution with preformed fibrils or by adding submicellar amounts of sodium dodecyl sulfate (SDS), where we obtain percentage standard deviations of 3-4% on the end ThT level. We conclude that there are multiple ways to achieve satisfactory levels of reproducibility, although the different conditions used to induce aggregation may lead to different fibrillization pathways.
[show abstract][hide abstract] ABSTRACT: Monomeric alpha-synuclein (alphaSN), which has no persistent structure in aqueous solution, is known to bind to anionic lipids with a resulting increase in alpha-helix structure. Here we show that at physiological pH and ionic strength, alphaSN incubated with different anionic lipid vesicles undergoes a marked increase in alpha-helical content at a temperature dictated either by the temperature of the lipid phase transition, or (in 1,2-DilauroylSN-Glycero-3-[Phospho-rac-(1-glycerol)] (DLPG), which is fluid down to 0 degrees C) by an intrinsic cold denaturation that occurs around 10-20 degrees C. This structure is subsequently lost in a thermal transition around 60 degrees C. Remarkably, this phenomenon is only observed for vesicles >100 nm in diameter and is sensitive to lipid chain length, longer chain lengths, and larger vesicles giving more cooperative unfolding transitions and a greater degree of structure. For both vesicle size and chain length, a higher degree of compressibility or permeability in the lipid thermal transition region is associated with a higher degree of alphaSN folding. Furthermore, the degree of structural change is strongly reduced by an increase in ionic strength or a decrease in the amount of anionic lipid. A simple binding-and-folding model that includes the lipid phase transition, exclusive binding of alphaSN to the liquid disordered phase, the thermodynamics of unfolding, and the electrostatics of binding of alphaSN to lipids is able to reproduce the two thermal transitions as well as the effect of ionic strength and anionic lipid. Thus the nature of alphaSN's binding to phospholipid membranes is intimately tied to the lipids' physico-chemical properties.
[show abstract][hide abstract] ABSTRACT: Intrinsic structural disorder is a prevalent feature of proteins with chaperone activity. Using a complementary set of techniques, we have structurally characterized LjIDP1 (intrinsically disordered protein 1) from the model legume Lotus japonicus, and our results provide the first structural characterization of a member of the Lea5 protein family (PF03242). Contrary to in silico predictions, we show that LjIDP1 is intrinsically disordered and probably exists as an ensemble of conformations with limited residual beta-sheet, turn/loop, and polyproline II secondary structure. Furthermore, we show that LjIDP1 has an inherent propensity to undergo a large conformational shift, adopting a largely alpha-helical structure when it is dehydrated and in the presence of different detergents and alcohols. This is consistent with an overrepresentation of order-promoting residues in LjIDP1 compared with the average of intrinsically disordered proteins. In line with functioning as a chaperone, we show that LjIDP1 effectively prevents inactivation of two model enzymes under conditions that promote protein misfolding and aggregation. The LjIdp1 gene is expressed in all L. japonicus tissues tested. A higher expression level was found in the root tip proximal zone, in roots inoculated with compatible endosymbiotic M. loti, and in functional nitrogen-fixing root nodules. We suggest that the ability of LjIDP1 to prevent protein misfolding and aggregation may play a significant role in tissues, such as symbiotic root nodules, which are characterized by high metabolic activity.
Journal of Biological Chemistry 10/2008; 283(45):31142-52. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Dendrimers are well-defined chemical polymers with a characteristic branching pattern that gives rise to attractive features such as antibacterial and antitumor activities as well as drug delivery properties. In addition, dendrimers can solubilize prion protein aggregates at very low concentrations, but their mode of action is unclear. We show that poly(propylene imine) dendrimers based on di-aminobutane (DAB) and modified with guanidinium surface groups reduce insulin thermostability and solubility considerably at microgram per microliter concentrations, while urea-modified groups have hardly any effect. Destabilization is markedly generation-dependent and is most pronounced for generation 3, which is also the most efficient at precipitating insulin. This suggests that proteins can interact with both dendrimer surface and interior. The pH-dependence reveals that interactions are mainly mediated by electrostatics, confirmed by studies on four other proteins. Ability to precipitate and destabilize are positively correlated, in contrast to conventional small-molecule denaturants and stabilizers, indicating that surface immobilization of denaturing groups profoundly affects its interactions with proteins.
[show abstract][hide abstract] ABSTRACT: We have previously described the complexity of the folding of the lipolytic enzyme cutinase from F. solani pisi in guanidinium chloride. Here we extend the refolding analysis by refolding from the pH-denatured state and analyze the folding behaviour in the presence of the weaker denaturant urea and the stronger denaturant guanidinium thiocyanate. In urea there is excellent consistency between equilibrium and kinetic data, and the intermediate accumulating at low denaturant concentrations is off-pathway. However, in GdmCl, refolding rates, and consequently the stability of the native state, vary significantly depending on whether refolding takes place from the pH- or GdmCl-denatured state, possibly due to transient formation of aggregates during folding from the GdmCl-denatured state. In GdmSCN, stability is reduced by several kcal/mol with significant aggregation in the unfolding transition region. The basis for the large variation in folding behaviour may be the denaturants' differential ability to support formation of exposed hydrophobic regions and consequent changes in aggregative properties during refolding.
Biochimica et Biophysica Acta 03/2007; 1774(2):323-33. · 4.66 Impact Factor