Alternative Assembly of α-Synuclein Leading to Protein Film Formation and Its Application for Developing Polydiacetylene-Based Sensing Materials.

Understanding the self-assembly process of amyloidogenic protein is valuable not only to find its pathological implication but also to prepare protein-based biomaterials. α-Synuclein (αS), a pathological component of Parkinson's disease, producing one-dimensional (1D) amyloid fibrils, has been employed to generate two-dimensional (2D) protein films by encouraging an alternative self-assembly process. At a high temperature of 50 °C, αS molecules self-assembled into 2D films instead of 1D amyloid fibrils, whereas the fibrils were the major product at 37 °C. Based on circular dichroism and Fourier transform infrared spectroscopy analyses, the film was produced via a structural transition from the initial random to still undefined but mostly the turn or loop structure, which was distinctive from the ß-sheet formation observed with the amyloid fibrils. The αS 2D film was also routinely prepared at the oil-water interface and used as a matrix to produce polydiacetylene-based sensing materials. 10,12-Pentacosadiynoic acids (PCDA) were aligned on the film and photopolymerized to form a π-conjugated molecular assembly yielding a blue color. Its colorimetric transition to red was induced by increasing the temperature. This functionalized protein film increased its height from 40 to 55 nm upon PCDA immobilization and exhibited enhanced physical and chemical stability. In addition, the modified film showed remarkably high electrical conductivity only in the red state. This film, therefore, can be considered as a robust protein-based hybrid biomaterial capable of simultaneously recognizing various external stimuli (heat, pH, and solvents) with changes in color and conductivity, and it is expected to be utilized as a basic material for the development of biocompatible sensors.

Morphological Evaluation of Meta-stable Oligomers of α-Synuclein with Small-Angle Neutron Scattering.

Amyloidogenesis of α-synuclein (αS) is considered to be a pathological phenomenon related to Parkinson's disease (PD). As a key component to reveal the fibrillation mechanism and toxicity, we have investigated an oligomeric species of αS capable of exhibiting the unit-assembly process leading to accelerated amyloid fibril formation. These oligomers previously shown to exist in a meta-stable state with mostly disordered structure and unable to seed the fibrillation were converted to either temperature-sensitive self-associative oligomers or NaCl-induced non-fibrillating oligomeric species. Despite their transient and disordered nature, the structural information of meta-stable αS oligomers (Meta-αS-Os) was successfully evaluated with small-angle neutron scattering (SANS) technique. By fitting the neutron scattering data with polydisperse Gaussian Coil (pGC) model, Meta-αS-O was analyzed as a sphere with approximate diameter of 100 Å. Its overall shape altered drastically with subtle changes in temperature between 37 °C and 43 °C, which would be responsible for fibrillar polymorphism. Based on their bifurcating property of Meta-αS-Os leading to either on-pathway or off-pathway species, the oligomers could be suggested as a crucial intermediate responsible for the oligomeric diversification and multiple fibrillation processes. Therefore, Meta-αS-Os could be considered as a principal target to control the amyloidogenesis and its pathogenesis.

Fabrication of Protease-Sensitive and Light-Responsive Microcapsules Encompassed with Single Layer of Gold Nanoparticles by Using Self-Assembly Protein of α-Synuclein.

A bioapplicable cargo delivery system requires the following characteristics of biocompatibility, in vivo stability, and selective cargo release at target sites. We introduce herein the microcapsules enclosed with a single-layered shell of gold nanoparticles (AuNPs) mutually connected by an amyloidogenic protein of α-synuclein (αS). The microcapsules were fabricated by producing oil(chloroform)-in-water Pickering emulsions of the αS-encapsulated AuNPs and subsequent molecular engagement of the outlying αS molecules, leading to formidable ß-sheet formation in the presence of chloroform. The wrinkled skin of microcapsules obtained after evaporation of the internal chloroform also reflects robustness of the protein-protein interaction, which was experimentally confirmed by their rheological stability. For the emulsions loaded with rhodamine 6G, their dye release was demonstrated to be controlled by proteases. Along with their photothermal activity, the AuNP-containing microcapsules and their proteolyzed fragments were therefore suggested to be capable of eliminating aberrant cells in the protease-activated pathologically affected areas. Orthogonal cargo loading was also achieved by encapsulating both hydrophobic and hydrophilic substances either directly dissolved in chloroform or prepackaged in inverted micelles, respectively. Microcapsule's functionality was further expanded by localizing quantum dots, magnetic nanoparticles, and antibodies inside or on the surface of the microcapsules. Taken together, these multimodal AuNP microcapsules are suggested to be an ideal cargo carrier system, which could be employed in not only biomedical theranostic applications as they exhibit structural robustness, specific targeting, triggered release, and photothermal activity but also sensor development in general.

EGCG-mediated Protection of the Membrane Disruption and Cytotoxicity Caused by the 'Active Oligomer' of α-Synuclein.

(-)-Epigallocatechin gallate (EGCG), the major component of green tea, has been re-evaluated with α-synuclein (αS), a pathological constituent of Parkinson's disease, to elaborate its therapeutic value. EGCG has been demonstrated to not only induce the off-pathway 'compact' oligomers of αS as suggested previously, but also drastically enhance the amyloid fibril formation of αS. Considering that the EGCG-induced amyloid fibrils could be a product of on-pathway SDS-sensitive 'transient' oligomers, the polyphenol effect on the transient 'active' oligomers (AOs) was investigated. By facilitating the fibril formation and thus eliminating the toxic AOs, EGCG was shown to suppress the membrane disrupting radiating amyloid fibril formation on the surface of liposomal membranes and thus protect the cells which could be readily affected by AOs. Taken together, EGCG has been suggested to exhibit its protective effect against the αS-mediated cytotoxicity by not only producing the off-pathway 'compact' oligomers, but also facilitating the conversion of 'active' oligomers into amyloid fibrils.

Molecular inscription of environmental information into protein suprastructures: temperature effects on unit assembly of α-synuclein oligomers into polymorphic amyloid fibrils.

Molecular-level storage of environmental information in biological structures in tangible forms, and their subsequent transfer to the next generation, has been studied using the phenomenon of amyloidogenesis, which defines a biochemical condition generating highly ordered protein aggregates known as amyloid fibrils. α-Synuclein oligomers shown to experience unit assembly as the formation of amyloid fibrils were used in the present study as an environment-sensing agent. With temperature varying in 2 °C intervals between 37 °C and 43 °C, the oligomeric unit assembly led to fibrillar polymorphism from a straight to a curly appearance, as assessed using TEM and small-angle neutron scattering; the different effects on the secondary structures were evaluated using attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy. The resulting diversified amyloid fibrils, which have distinctive molecular characteristics, were shown to be inherited by the next generation through the self-propagating property of amyloidogenesis. Storage of intangible temperature information in the diversified protein suprastructures and perpetuation of the stored information in the form of polymorphic amyloid fibrils could represent molecular inscription of environmental information into biological systems; this could further extend our understanding of any physiological/pathological significance of amyloidogenic polymorphism and be utilized in the area of nanobiotechnology to process various external signals.

Radiating amyloid fibril formation on the surface of lipid membranes through unit-assembly of oligomeric species of α-synuclein.

BACKGROUND: Lewy body in the substantia nigra is a cardinal pathological feature of Parkinson's disease. Despite enormous efforts, the cause-and-effect relationship between Lewy body formation and the disorder is yet to be explicitly unveiled. METHODOLOGY/PRINCIPAL FINDINGS: Here, we showed that radiating amyloid fibrils (RAFs) were instantly developed on the surface of synthetic lipid membranes from the ß-sheet free oligomeric species of α-synuclein through a unit-assembly process. The burgeoning RAFs were successfully matured by feeding them with additional oligomers, which led to concomitant dramatic shrinkage and disintegration of the membranes by pulling off lipid molecules to the extending fibrils. Mitochondria and lysosomes were demonstrated to be disrupted by the oligomeric α-synuclein via membrane-dependent fibril formation. CONCLUSION: The physical structure formation of amyloid fibrils, therefore, could be considered as detrimental to the cells by affecting membrane integrity of the intracellular organelles, which might be a molecular cause for the neuronal degeneration observed in Parkinson's disease.

Amyloid hydrogel derived from curly protein fibrils of alpha-synuclein.

Elucidation of molecular assembly mechanism of protein-based suprastructure formation is pivotal to develop biomaterials. A single amyloidogenic protein of alpha-synuclein turned into two morphologically distinctive amyloid fibrils - 'curly' (CAF) vs. 'straight' (SAF) - depending on its fibrillation processes. Mutually exclusive production of CAF and SAF was achieved with either centrifugal membrane filtration of the preformed oligomeric species of alpha-synuclein or agitated incubation of its monomeric form, representing amyloidogeneses via double-concerted and nucleation-dependent fibrillation model, respectively. Differences in secondary structures of CAF and SAF have been suggested to be responsible for their morphological uniqueness with structural flexibility and mechanical strength. Both polymorphs exerted the self-propagation property, demonstrating that their characteristic morphologies were inherited for two consecutive generations to daughter and granddaughter fibrils through the seed-dependent fibrillation procedure. Accumulation of CAF produced amyloid hydrogel composed of fine nano-scaled three-dimensional protein fibrillar network. The hydrogel made of daughter CAF was demonstrated to be a suitable nanomatrix for enzyme entrapment, which protected the entrapped enzyme of horseradish peroxidase from loss of activity due to multiple catalyses and heat treatment. The nano-scaled fibrillar network of CAF, therefore, could exhibit a full potential to be further applied in the promising areas of nanobiotechnology including tissue engineering, drug delivery, nanofiltration and biosensor development.