Ghibom Bhak

Granular Assembly of α-Synuclein Leading to the Accelerated Amyloid Fibril Formation with Shear Stress

α-Synuclein participates in the Lewy body formation of Parkinsons disease. Elucidation of the underlying molecular mechanism of the amyloid fibril formation is crucial not only to develop a controlling strategy toward the disease, but also to apply the protein fibrils for future biotechnology. Discernable homogeneous granules of α-synuclein composed of approximately 11 monomers in average were isolated in the middle of a lag phase during the in vitro fibrillation process. They were demonstrated to experience almost instantaneous fibrillation during a single 12-min centrifugal membrane-filtration at 14,000×g. The granular assembly leading to the drastically accelerated fibril formation was demonstrated to be a result of the physical influence of shear force imposed on the preformed granular structures by either centrifugal filtration or rheometer. Structural rearrangement of the preformed oligomomeric structures is attributable for the suprastructure formation in which the granules act as a growing unit for the fibril formation. To parallel the prevailing notion of nucleation-dependent amyloidosis, we propose a double-concerted fibrillation model as one of the mechanisms to explain the in vitro fibrillation of α-synuclein, in which two consecutive concerted associations of monomers and subsequent oligomeric granular species are responsible for the eventual amyloid fibril formation.

Instantaneous Amyloid Fibril Formation of α-Synuclein from the Oligomeric Granular Structures in the Presence of Hexane

Amyloid fibrils found in various neurodegenerative disorders are also recognized as high-performance protein nanomaterials with a formidable rigidity. Elucidation of an underlying molecular mechanism of the amyloid fibril formation is crucial not only to develop controlling strategy toward the diseases, but also to apply the protein fibrils for future nanobiotechnology. alpha-Synuclein is an amyloidogenic protein responsible for the radiating filament formation within Lewy bodies of Parkinsons disease. The amyloid fibril formation of alpha-synuclein has been shown to be induced from the oligomeric granular species of the protein acting as a growing unit by experiencing structural rearrangement within the preformed oligomeric structures in the presence of an organic solvent of hexane. This granule-based concerted amyloid fibril formation model would parallel the prevalent notion of nucleation-dependent fibrillation mechanism in the area of amyloidosis.

Amyloid Fibrillar Meshwork Formation of Iron‐Induced Oligomeric Species of Aβ40 with Phthalocyanine Tetrasulfonate and Its Toxic Consequences

Amyloid-b protein (Ab) is a pathological component of Alzheimer’s disease (AD) that participates in the senile plaque formation in the neocortex of the patient’s brain. Ab is generated from the ubiquitous amyloid precursor protein (APP) through proteolysis by b and g-secretases. Abnormal accumulation of Ab is believed to cause the neuronal degeneration that is selective to the cholinergic neurons in particular. Selfassociation of Ab leads to various fibrillation states, such as soluble high-molecular-weight oligomeric forms, globular species, which are known as Ab-derived diffusible ligands, protofibrils as metastable intermediates, and finally amyloid fibrils. 6] Although the toxic species has not been defined unequivocally, the prefibrillar assemblies of Ab are considered to be responsible for the neuronal degeneration by affecting the stability of biological membranes. Besides this Ab-only hypothesis of exert neurotoxicity, metals have been also suggested to act as an additional toxic component within the Ab amyloids. Those metals, including iron, copper, and zinc would provide a pivotal center for reactive oxygen species (ROS) generation at the expense of endogenous reducing agents. For treatment strategies of AD, therefore, suppression of Ab production, clearance of the toxic prefibillar species with antiAb antibodies, antioxidants, and metal-chelating agent have been extensively tested. Because the neurodegeneration that is observed in AD is induced from multifactorial causes, a novel therapeutic strategy that concomitantly handles some of the multiple causal candidates at the same time is highly recommendable. We herein introduce phthalocyanine tetrasulfonate (PcTS) as a cyclic tetrapyrrole compound with a planar and hydrophobic central aromatic macrocyclic structure, which accommodates various metal ions. Metal-containing PcTS tends to self-assemble into nanoparticles, monolayers, and thin films. By taking advantage of those properties of metal chelation and molecular self-assembly, PcTS has been employed not only to remove the redox-active metals from the toxic metal-induced oligomeric species of Ab40 but also to convert the protofibrillar species into an amyloid fibrillar meshwork if possible (Scheme 1). This dual function of PcTS, therefore, could take care of the toxic consequences of the prefibrillar species due to the membrane destabilization by the oligomeric structures

Free-Standing Gold-Nanoparticle Monolayer Film Fabricated by Protein Self-Assembly of α-Synuclein†

Free-standing nanoparticle films are of great importance for developing future nano-electronic devices. We introduce a protein-based fabrication strategy of free-standing nanoparticle monolayer films. α-Synuclein, an amyloidogenic protein, was utilized to yield a tightly packed gold-nanoparticle monolayer film interconnected by protein β-sheet interactions. Owing to the stable protein-protein interaction, the film was successfully expanded to a 4-inch diameter sheet, which has not been achieved with any other free-standing nanoparticle monolayers. The film was flexible in solution, so it formed a conformal contact, surrounding even microspheres. Additionally, the monolayer film was readily patterned at micrometer-scale and thus unprecedented double-component nanoparticle films were fabricated. Therefore, the free-floating gold-nanoparticle monolayer sheets with these properties could make the film useful for the development of bio-integrated nano-devices and high-performance sensors.

Controlled Charge Trapping and Retention in Large-Area Monodisperse Protein Metal-Nanoparticle Conjugates

Here, we report on charge-retention transistors based on novel protein-mediated Au nanoparticle (NP) arrays, with precise control over dimension and distribution. Individual NPs are coated with alpha-synuclein, an amyloidogenic protein responsible for Lewy body formation in Parkinsons disease. Subsequently, a monolayer of protein-NP conjugates is successfully created via a simple and scalable solution deposition to function as distributed nanoscale capacitors. Controllability over the film structure translates into the tunability of the electrical performance; pentacene-based organic transistors feature widely varying programmability and relaxation dynamics, providing versatility for various unconventional memory applications.

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.

κ-Casein-based hierarchical suprastructures and their use for selective temporal and spatial control over neuronal differentiation.

Functions are diversified by producing hierarchical structures from a single raw material. Biologically compatible milk protein of κ-casein has been employed to fabricate higher-order suprastructures. In the presence of dithiothreitol and heat treatment, κ-casein transforms into amyloid fibrils with distinctive morphology attributable to mechanism-based fibrillar polymorphism. As the fibrils elongate to yield high aspect ratio during high-temperature incubation, the resulting fibrils laterally associate into the liquid crystalline state by forming a two-dimensional fibrillar array. Following a desalting process, the fibrillar arrays turn into a three-dimensional matrix of hydrogel that could be selectively disintegrated by subsequent salt treatment. The hydrogel was demonstrated to be a matrix capable of exhibiting controlled release of bioactive substances like retinoic acid, which led to temporal and spatial control over the differentiation of neuronal cells. Therefore, the hierarchical suprastructure formation derived from the single protein of κ-casein producing one-dimensional protein nanofibrils, a two-dimensional liquid crystalline state and a three-dimensional hydrogel could be widely appreciated in various areas of nanobiotechnology including drug delivery and tissue engineering.

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

Abstract(−)-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.

Photoelectric Protein Nanofibrils of α-Synuclein with Embedded Iron and Phthalocyanine Tetrasulfonate†

The development of photoelectric systems that respond to visible light has gained a great deal of attention for the preparation of light-harvesting and optical-switching systems, which would contribute to the construction of sensors, electronics, optical information processing systems, and energy-transducing devices. Porphyrinic molecules are considered to be ideal for the photoinduced electron-transfer process owing to their excellent photonic, electronic, and catalytic properties. To harness these properties in a more effective and practical way, their hierarchical suprastructures have been engineered to give rise to controlled spatial arrangement of the unique aromatic compounds by taking advantage of their self-interactive property. Herein, we demonstrate selective localization of the porphyrinic compound phthalocyanine tetrasulfonate (PcTS) into a protein matrix of amyloid fibrils made of a-synuclein in the presence of iron, which results in conversion of the dielectric protein nanofibrils into light-inducible conductive hybrid nanomaterials.

High-Density Single-Layer Coating of Gold Nanoparticles onto Multiple Substrates by Using an Intrinsically Disordered Protein of α-Synuclein for Nanoapplications

Functional graffiti of nanoparticles onto target surface is an important issue in the development of nanodevices. A general strategy has been introduced here to decorate chemically diverse substrates with gold nanoparticles (AuNPs) in the form of a close-packed single layer by using an omni-adhesive protein of α-synuclein (αS) as conjugated with the particles. Since the adsorption was highly sensitive to pH, the amino acid sequence of αS exposed from the conjugates and its conformationally disordered state capable of exhibiting structural plasticity are considered to be responsible for the single-layer coating over diverse surfaces. Merited by the simple solution-based adsorption procedure, the particles have been imprinted to various geometric shapes in 2-D and physically inaccessible surfaces of 3-D objects. The αS-encapsulated AuNPs to form a high-density single-layer coat has been employed in the development of nonvolatile memory, fule-cell, solar-cell, and cell-culture platform, where the outlying αS has played versatile roles such as a dielectric layer for charge retention, a sacrificial layer to expose AuNPs for chemical catalysis, a reaction center for silicification, and biointerface for cell attachment, respectively. Multiple utilizations of the αS-based hybrid NPs, therefore, could offer great versatility to fabricate a variety of NP-integrated advanced materials which would serve as an indispensable component for widespread applications of high-performance nanodevices.