Dataset Information

Unveiling a Selective Mechanism for the Inhibition of ?-Synuclein Aggregation by ?-Synuclein.

ABSTRACT: ?-Synuclein (?S) is an intrinsically disordered protein that is associated with Parkinson's disease (PD) through its ability to self-assemble into oligomers and fibrils. Inhibition of this oligomerization cascade is an interesting approach to developing therapeutical strategies and ?-synuclein (?S) has been described as a natural negative regulator of this process. However, the biological background and molecular mechanisms by which this inhibition occurs is unclear. Herein, we focused on assessing the effect of ?S on the aggregation of five ?S pathological mutants linked to early-onset PD (A30P, E46K, H50Q, G51D and A53T). By coupling single molecule fluorescence spectroscopy to a cell-free protein expression system, we validated the ability of ?S to act as a chaperone of ?S, effectively inhibiting its aggregation. Interestingly, we found that ?S does so in a selective manner, i.e., is a more effective inhibitor for certain ?S pathological mutants-A30P and G51D-as compared to E46K, H50Q and A53T. Moreover, two-color coincidence experiments proved that this discrepancy is due to a preferential incorporation of ?S into smaller oligomers of ?S. This was validated by showing that the chaperoning effect was lost when proteins were mixed after being expressed individually. This study highlights the potential of fluorescence spectroscopy to deconstruct ?S aggregation cascade and its interplay with ?S.

SUBMITTER: Leitao A

PROVIDER: S-EPMC5855556 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Publications

Unveiling a Selective Mechanism for the Inhibition of α-Synuclein Aggregation by β-Synuclein.

Leitao Andre A Bhumkar Akshay A Hunter Dominic J B DJB Gambin Yann Y Sierecki Emma E

International journal of molecular sciences 20180124 2

α-Synuclein (αS) is an intrinsically disordered protein that is associated with Parkinson's disease (PD) through its ability to self-assemble into oligomers and fibrils. Inhibition of this oligomerization cascade is an interesting approach to developing therapeutical strategies and β-synuclein (βS) has been described as a natural negative regulator of this process. However, the biological background and molecular mechanisms by which this inhibition occurs is unclear. Herein, we focused on assess ...[more]

PMID: 29364143

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Project description:While several polyphenols were found to either inhibit or modulate the aggregation of proteins implicated in neurodegenerative diseases, such as Parkinson's disease (PD), discrepant action mechanisms have been reported. This, in addition to some polyphenols' pan-assay interference compounds' reputation, casts some doubts concerning their therapeutic relevance. Here, we studied, through molecular dynamics and enhanced sampling methods, the aggregation of 11-mer peptides from the non-amyloid-β component, an aggregation-prone domain of α-synuclein (α-syn) implicated in PD and other synucleinopathies, in neat water and aqueous solutions of resveratrol (RSV) and gallic acid (GA). Further, simulations of the complete protein were carried out in aqueous urea, RSV, and GA solutions. Our results show that peptide aggregation is not disrupted by either phenolic compound. Thus, instead, intrusion of RSV and GA in the inter-peptide region induces a peptide-peptide re-orientation, favoring terminal interactions that manifest in the formation of barrierless solvent-separated configurations. Moreover, although the (poly)phenols induce a pronounced peptide dewetting at high concentrations, β-sheet-rich regions, a hallmark of α-syn aggregation, are not disrupted. Thus, our results indicate that, if anything, RSV and GA delay or modulate peptide aggregation at high concentrations via the stabilization of solvent-separated conformations as opposed to aggregation inhibition. Structural analysis of the full protein, however, shows that the (poly)phenols induce more extended conformations of α-syn, similar to urea, possibly also influencing its aggregation propensity. However, opposite to urea, the (poly)phenols reduce α-syn's conformational space, likely due to steric effects and a slowdown of the solvent dynamics. These effects are concentration-dependent and possibly unattainable at therapeutic-relevant concentrations. These results suggest that the aggregation inhibition activity of RSV and GA in vitro should involve, instead, either the non-covalent binding to oligomeric intermediates or the stabilization of the monomer and/or oligomers through the formation of covalent bonds of the respective quinones with α-syn. In addition, the enhanced aggregation tendency of the peptides observed here could be associated with the formation of non-toxic oligomers, reported for some polyphenols.

Project description:Aggregation of neuronal protein ?-synuclein leads to the formation of amyloid fibrils, which are associated with the development of Parkinson's disease. The mechanism of ?-synuclein pathology is not fully understood and is a subject of active research in the field. To tackle this problem, the fusions of fluorescent proteins to ?-synuclein C-terminus are often used in cellular and animal studies. The effects induced by such ?-synuclein sequence extension on ?-synuclein aggregation propensity are, however, not systematically examined despite the evidence that the negatively charged C-terminus plays a critical role in the regulation of ?-synuclein aggregation. In this work, we investigated how the charge and length variations of the C-terminus affect the aggregation propensity of ?-synuclein. To address these questions, we prepared mutants of ?-synuclein carrying additional moieties of different charge and length at the protein C-terminus. We determined the rates of two different aggregation stages (primary nucleation and elongation) based on a thioflavin T kinetic assay. We observed that all mutants bearing neutrally charged moieties of different length fibrilized slower than wild-type ?-synuclein. The primary nucleation and elongation rates strongly decreased with increase of the C-terminal extension length. Meanwhile, charge variation of the C-terminus significantly changed the rate of ?-synuclein nucleation, but did not markedly affect the rate of fibril elongation. Our data demonstrate that both the charge and length of the C-terminus play an important role at the stage of initial fibril formation, but the stage of fibril elongation is affected mainly by the length of C-terminal extension. In addition, our results suggest that there are at least two steps of incorporation of ?-synuclein monomers into the amyloid fibril: namely, the initial monomer binding to the fibril end (charge-dependent, relatively fast), and the subsequent conformational change of the protein (charge-independent, relatively slow, and thus the rate-limiting step).

Dataset Information

Unveiling a Selective Mechanism for the Inhibition of ?-Synuclein Aggregation by ?-Synuclein.

Publications

Unveiling a Selective Mechanism for the Inhibition of α-Synuclein Aggregation by β-Synuclein.

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