Rapid Anionic Micelle-mediated α-Synuclein Fibrillization in Vitro

161

171

Because oligomers and aggregates of the protein α-synuclein (αS) are implicated in the initiation and progression of Parkinson's disease, investigation of various αS aggregation pathways and intermediates aims to clarify the etiology of this common neurodegenerative disorder. Here, we report the formation of short, flexible, β-sheet-rich fibrillar species by incubation of αS in the presence of intermediate (10-20% v∕v) concentrations of 2,2,2-trifluoroethanol (TFE). We find that efficient production of these TFE fibrils is strongly correlated with the TFE-induced formation of a monomeric, partly helical intermediate conformation of αS, which exists in equilibrium with the natively disordered state at low [TFE] and with a highly α-helical conformation at high [TFE]. This partially helical intermediate is on-pathway to the TFE-induced formation of both the highly helical monomeric conformation and the fibrillar species. TFE-induced conformational changes in the monomer protein are similar for wild-type αS and the C-terminal truncation mutant αS1-102, indicating that TFE-induced structural transitions involve the N terminus of the protein. Moreover, the secondary structural transitions of three Parkinson's disease-associated mutants, A30P, A53T, and E46K, are nearly identical to wild-type αS, but oligomerization rates differ substantially among the mutants. Our results add to a growing body of evidence indicating the involvement of helical intermediates in protein aggregation processes. Given that αS is known to populate both highly and partially helical states upon association with membranes, these TFE-induced conformations imply relevant pathways for membrane-induced αS aggregation both in vitro and in vivo.is one of a number of synucleopathies in which aggregation of the protein α-Synuclein (αS) is linked to pathogenesis (1). αS is intrinsically disordered, but in the presence of lipid or detergent vesicles or micelles, adopts a highly helical structure in which its N-terminal region is membrane-bound and the C-terminal tail remains predominantly free and unstructured (2, 3). Although most PD cases are sporadic or idiopathic, three point mutations of α-Synuclein-A53T, A30P, and E46K-are associated with familial and early-onset disease (see Review (4)).In addition to its free and membrane-bound states, αS adopts partially structured intermediate conformations under low-pH or high-temperature conditions (5). A folding intermediate has also been detected at low [TFE] (6). Conditions favoring the formation of these intermediates also promote amyloid fibril growth, possibly implicating intermediate conformers as key species in the aggregation pathways.Here, we examine TFE-induced monomer conformational changes, oligomerization, and fibrillization in detail for wild-type (WT) αS, C terminally truncated WT αS (αS102), and the PDassociated αS mutants A30P, A53T, and E46K, expanding upon previous studies by Munishkina, et. al. (6) and Li, et. al. (7). This research also complements our previous fluorescence correlatio...

Section: Discussionmentioning

confidence: 91%

Identification of a helical intermediate in trifluoroethanol-induced alpha-synuclein aggregation

Anderson

Ramlall

Rospigliosi

et al. 2010

161

171

“…Besides studying the structural basis for the putative physiological functions of ␣-synuclein, SDSL could also be used to investigate the conformational changes involved in the transition from unfolded or helical synuclein into toxic oligomeric forms (40). In this respect, it is interesting to note that, at least under some conditions, membrane interaction can facilitate the formation of such oligomers (41)(42)(43) and that modulation of the membrane interaction could be responsible for the formation of toxic oligomers in vivo (44,45). Thus, the molecular understanding of the helical, membrane-bound form discussed here might prove to be an important starting point for our understanding of the misfolding that occurs in disease.…”

Section: Discussionmentioning

confidence: 99%

“…Inasmuch as a continuous ␣-helix of 90 or more amino acids would be at least 130 Å long, it would have to almost completely wrap around SDS micelles, but span only Ϸ20% of the small unilamellar vesicle circumference. The curvature strain of SDS micelles might therefore require breakage of the elongated helix near its midpoint (residues [42][43][44].…”

Section: Discussionmentioning

confidence: 99%

Structure of membrane-bound α-synuclein studied by site-directed spin labeling

Jao

Der-Sarkissian²,

Chen³

et al. 2004

346

398

Many of the proposed physiological functions of ␣-synuclein, a protein involved in the pathogenesis of Parkinson's disease, are related to its ability to interact with phospholipids. To better understand the conformational changes that occur upon membrane binding of monomeric ␣-synuclein, we performed EPR analysis of 47 singly labeled ␣-synuclein derivatives. We show that membrane interaction is mediated by major conformational changes within seven N-terminal 11-aa repeats, which reorganize from a highly dynamic structure into an elongated helical structure devoid of significant tertiary packing. Furthermore, we find that analogous positions from different repeats are in equivalent locations with respect to membrane proximity. These and other findings suggest a curved membrane-dependent ␣-helical structure, wherein each 11-aa repeat takes up three helical turns. Similar helical structures could also apply to apolipoproteins and other lipid-interacting proteins with related 11-aa repeats.T he protein ␣-synuclein is the main component of Lewy bodies, a class of intracellular inclusions that is highly characteristic in Parkinson's disease (PD) (1). A causative role of ␣-synuclein in PD has been supported by genetic studies of familial forms of this disease (2-4) as well as by various animal models (5). In addition to its involvement in PD, ␣-synuclein may also play important roles in Alzheimer's disease, dementia with Lewy bodies, multiple system atrophy, and Hallervorden-Spatz syndrome (6, 7).Although not yet fully understood, the physiological function of ␣-synuclein is likely to involve a role in modulating synaptic plasticity (8), presynaptic vesicle pool size, and neurotransmitter release (9-11), as well as vesicle recycling (12). In agreement with these membrane-related functions, ␣-synuclein has been shown to interact with liposomes in vitro (13-16). According to circular dichroism analysis, this interaction causes ␣-synuclein to undergo a conformational change from an unstructured monomer in solution (13,17,18) to an ␣-helical, membrane-bound protein. Based upon sequence analysis, it was recognized early on that the N-terminal portion of ␣-synuclein was likely to mediate lipid interaction (8,13). The N terminus of ␣-synuclein contains seven repeats, each of which is made up of 11 aa (Fig. 1). These repeats are similar to those found in apolipoproteins, and it was proposed that the lipid interaction of ␣-synuclein could be similar to that of the apolipoproteins (8, 13). The involvement of the N terminus in membrane interaction was subsequently confirmed experimentally by analysis of ␣-synuclein deletion mutants (15) and NMR studies of liposomebound ␣-synuclein (18-20). The latter studies revealed an ordering of the N-terminal repeat regions induced upon membrane binding whereas the highly charged C terminus remained unstructured and, therefore, was not involved in membrane interaction. Beyond these data, however, direct structural information, such as the precise location, length, orientation, and number of...

“…Previous work has shown that the process of nucleation of α-synuclein amyloid fibrils is likely to be heterogeneous and catalyzed by environmental features, such as air-water interfaces (26,27), lipid bilayers (28), SDS micelles and other anionic surfactants (11,29,30), or artificial interfaces such as the coatings of containers or stir bars (31). In addition, mechanical action, such as shaking and stirring, is often used to accelerate the aggregation of α-synuclein (32).…”

Section: Primary Nucleation and Fragmentation Can Be Selectively Enhamentioning

confidence: 99%

Solution conditions determine the relative importance of nucleation and growth processes in α-synuclein aggregation

Buell

Galvagnion

Gaspar³

et al. 2014

603

867

The formation of amyloid fibrils by the intrinsically disordered protein α-synuclein is a hallmark of Parkinson disease. To characterize the microscopic steps in the mechanism of aggregation of this protein we have used in vitro aggregation assays in the presence of preformed seed fibrils to determine the molecular rate constant of fibril elongation under a range of different conditions. We show that α-synuclein amyloid fibrils grow by monomer and not oligomer addition and are subject to higher-order assembly processes that decrease their capacity to grow. We also find that at neutral pH under quiescent conditions homogeneous primary nucleation and secondary processes, such as fragmentation and surface-assisted nucleation, which can lead to proliferation of the total number of aggregates, are undetectable. At pH values below 6, however, the rate of secondary nucleation increases dramatically, leading to a completely different balance between the nucleation and growth of aggregates. Thus, at mildly acidic pH values, such as those, for example, that are present in some intracellular locations, including endosomes and lysosomes, multiplication of aggregates is much faster than at normal physiological pH values, largely as a consequence of much more rapid secondary nucleation. These findings provide new insights into possible mechanisms of α-synuclein aggregation and aggregate spreading in the context of Parkinson disease.seeding | prion-like behavior | neurodegenerative disease | kinetic analysis | electrostatic interactions T he conversion of soluble peptide and protein molecules into insoluble amyloid fibrils is of great interest in fields of science ranging from molecular medicine to nanotechnology (1). The formation of amyloid fibrils is a characteristic feature of a substantial number of increasingly common medical disorders, including neurodegenerative conditions such as Alzheimer's and Parkinson diseases (2). Elucidating the fundamental mechanistic steps involved in the conversion from the soluble to the fibrillar forms of the peptides and proteins involved in such disorders is crucial for understanding their origin and proliferation, and hence for exploring in a rational manner new and effective therapeutic strategies through which to combat their onset or progression (3).One general aspect of amyloid diseases is that once the first aggregates are formed it is very difficult to stop or reverse the aggregation process. This implies that aggregation needs to be studied in both the absence and presence of preformed aggregates, commonly known as seeds, to deepen our understanding of the mechanism of the self-assembly process in vivo. It is possible to define from in vitro studies the rate constants for the multiplicity of microscopic steps that increase the number and total mass of the different types of aggregates that are populated during this process. Such an analysis has recently been carried out for the Aβ42 peptide (4) associated with Alzheimer's disease by combining experimental and theoretical methodolo...