Fabrication of a Dual Stimuli-Responsive Assorted Film Comprising Magnetic- and Gold-Nanoparticles with a Self-Assembly Protein of α-Synuclein.

Development of sensing elements for controllable soft materials is crucial to improve their responsiveness toward remotely provided external stimuli. Magnetic nanoparticles (MNPs) and gold nanoparticles (AuNPs) have been coassembled into a flexible free-floating 2D film to produce a shape deformable mobile structure in the presence of magnetic field and light irradiation by employing a self-assembly protein of α-synuclein (αS). αS was demonstrated to be essential for the preparation of a multisensory system because the intrinsically disordered protein led to a complete dispersion of MNPs to an average size of 10 nm in aqueous solution, pH-dependent closely packed single layer adsorption of αS-MNPs, and α-helix-mediated free-floating MNP monolayer film formation upon dissolving the underlying polycarbonate substrate with chloroform. As AuNPs were incorporated into the assorted hybrid film in the presence of MNPs, however, the ß-sheet component became prominent. By placing the assorted film between a spin-coated thin layer of thermoresponsive P(AAc-co-NIPAAm) hydrogel comprising acrylic acid and N-isopropylacrylamide and a passive layer of silicone elastomer, the resulting triply structure exhibited not only magnet-induced locomotion but also shape deformation due to asymmetric contraction of the sandwiching two layers caused by the heat generated by AuNPs upon near IR irradiation. In fact, two adjoining planar layers of another triply structure were shown to form a three-dimensional lotus flower with the light. This multisensory system is suggested to be further functionalized by modifying the αS molecules and incorporating additional nanoparticles to react to diverse stimuli, which would make the system be utilized in the areas of not only soft robotics but also foldable electronics, high-performance sensors/actuators, and medical/wearable applications.

Parkinson's disease-related phosphorylation at Tyr39 rearranges α-synuclein amyloid fibril structure revealed by cryo-EM.

Posttranslational modifications (PTMs) of α-synuclein (α-syn), e.g., phosphorylation, play an important role in modulating α-syn pathology in Parkinson's disease (PD) and α-synucleinopathies. Accumulation of phosphorylated α-syn fibrils in Lewy bodies and Lewy neurites is the histological hallmark of these diseases. However, it is unclear how phosphorylation relates to α-syn pathology. Here, by combining chemical synthesis and bacterial expression, we obtained homogeneous α-syn fibrils with site-specific phosphorylation at Y39, which exhibits enhanced neuronal pathology in rat primary cortical neurons. We determined the cryo-electron microscopy (cryo-EM) structure of the pY39 α-syn fibril, which reveals a fold of α-syn with pY39 in the center of the fibril core forming an electrostatic interaction network with eight charged residues in the N-terminal region of α-syn. This structure composed of residues 1 to 100 represents the largest α-syn fibril core determined so far. This work provides structural understanding on the pathology of the pY39 α-syn fibril and highlights the importance of PTMs in defining the polymorphism and pathology of amyloid fibrils in neurodegenerative diseases.

Chemoenzymatic Semisynthesis of Phosphorylated α-Synuclein Enables Identification of a Bidirectional Effect on Fibril Formation.

Post-translational modifications (PTMs) impact the pathological aggregation of α-synuclein (αS), a hallmark of Parkinson's disease (PD). Here, we synthesize αS phosphorylated at tyrosine 39 (pY39) through a novel route using in vitro enzymatic phosphorylation of a fragment followed by ligation to form the full-length protein. We can execute this synthesis in combination with unnatural amino acid mutagenesis to include two fluorescent labels for Förster resonance energy transfer (FRET) studies. We determine the effect of pY39 on the aggregation of αS and compare our authentically phosphorylated material to the corresponding glutamate 39 "phosphomimetic." Intriguingly, we find that αS-pY39 can either accelerate or decelerate aggregation, depending on the fraction of phosphorylated protein. The αS-E39 mutant can qualitatively reproduce some, but not all, of these effects. FRET measurements and analysis of existing structures of αS help to provide an explanation for this phenomenon. Our results have important implications for the treatment of PD patients with tyrosine kinase inhibitors and highlight the importance of validating phosphomimetics through studies of authentic PTMs.

Conformation change of α-synuclein(61－95) at the air-water interface and quantitative measurement of the tilt angle of the axis of its α-helix by multiple angle incidence resolution spectroscopy.

Various techniques have been developed to determine protein's structure to understand how proteins work.  Compared with X-ray crystallography requiring proteins to form single crystal structure and NMR which usually needs long time measurement, surface FT-IR techniques are able to quickly determine the tilt angle (the key information to determine whether the α-helix is transmembrane) of peptides/proteins in a monolayer at the interface (e.g. membranes). Specifically, for α-helical peptides/proteins in membrane, the tilt angle of the axis is one of the key information. In this paper, Multiple Angle Incidence Resolution Spectroscopy (MAIRS), a recently developed surface FTIR technique, was applied for the first time to quantitatively determine the tilt angle of the axis of α-helical model peptide related to α-synuclein (α-syn). α-Syn is a 140-amino-acid presynaptic protein whose aggregation is the hallmark of Parkinson's disease (PD). It is difficult for α-syn to form a single crystal structure and the primary structure of α-syn constitutes three domains: the N-terminus containing residues 1-60; the nonamyloid component (NAC) which spans residues 61-95 and is highly prone to aggregation; and C-terminus with residues 96-140. Here, the NAC part (i.e., α-syn(61-95)) responsible for the aggregation was found to change its unstructured conformation in aqueous solution to α-helix at the air-water interface by circular dichroism and MAIRS. In addition, the instinct power of MAIRS to quantitatively measure the tilt angle of the axis of α-helical α-syn(61-95) in monolayer was fully exhibited. Therefore, MAIRS is a potential supplemental technique to X-ray crystallography and NMR to determine the structure of membrane peptides/proteins.

Mechanistic Understanding of the Interactions between Nano-Objects with Different Surface Properties and α-Synuclein.

Aggregation of the natively unfolded protein α-synuclein (α-syn) is key to the development of Parkinson's disease (PD). Some nanoparticles (NPs) can inhibit this process and in turn be used for treatment of PD. Using simulation strategies, we show here that α-syn self-assembly is electrostatically driven. Dimerization by head-to-head monomer contact is triggered by dipole-dipole interactions and subsequently stabilized by van der Waals interactions and hydrogen bonds. Therefore, we hypothesized that charged nano-objects could interfere with this process and thus prevent α-syn fibrillation. In our simulations, positively and negatively charged graphene sheets or superparamagnetic iron oxide NPs first interacted with α-syn's N/C terminally charged residues and then with hydrophobic residues in the non-amyloid-ß component (61-95) region. In the experimental setup, we demonstrated that the charged nano-objects have the capacity not only to strongly inhibit α-syn fibrillation (both nucleation and elongation) but also to disaggregate the mature fibrils. Through the α-syn fibrillation process, the charged nano-objects induced the formation of off-pathway oligomers.

Vibrational Circular Dichroism Reveals Supramolecular Chirality Inversion of α-Synuclein Peptide Assemblies upon Interactions with Anionic Membranes.

Parkinson's disease is an incurable neurodegenerative disorder caused by the aggregation of α-synuclein (AS). This amyloid protein contains a 12-residue-long segment, AS71-82, that triggers AS pathological aggregation. This peptide is then essential to better understand the polymorphism and the dynamics of formation of AS fibrillar structures. In this work, vibrational circular dichroism showed that AS71-82 is random coil in solution and forms parallel ß-sheet fibrillar aggregates in the presence of anionic vesicles. Vibrational circular dichroism, with transmission electronic microscopy, revealed that the fibrillar structures exhibit a nanoscale tape-like morphology with a preferential supramolecular helicity. Whereas the structure handedness of some other amyloid peptides has been shown to be driven by pH, that of AS71-82 is controlled by peptide concentration and peptide-to-lipid (P:L) molar ratio. At low concentrations and low P:L molar ratios, AS71-82 assemblies have a left-twisted handedness, whereas at high concentrations and high P:L ratios, a right-twisted handedness is adopted. Left-twisted assemblies interconvert into right-twisted ones with time, suggesting a maturation of the amyloid structures. As fibril species with two chiralities have also been reported previously in Parkinson's disease Lewy bodies and fibrils, the present results seem relevant to better understand AS amyloid assembly and fibrillization in vivo. From a diagnosis or therapeutic point of view, it becomes essential that future fibril probes, inhibitors, or breakers target pathological assemblies with specific chirality and morphology, in particular, because they may change with the stage of the disease.

Exploring the Roles of Post-Translational Modifications in the Pathogenesis of Parkinson's Disease Using Synthetic and Semisynthetic Modified α-Synuclein.

Alpha-synuclein (α-syn), a small soluble protein containing 140 amino acids, is associated with the recycling pool of synaptic vesicles in presynaptic terminals. The misfolding and aggregation of α-syn is closely related to a group of neurodegenerative diseases, including Parkinson's disease (PD), which is one of the most common progressive neurodegenerative diseases. Varieties of the post-translational modifications (PTMs) of α-syn, including phosphorylation, ubiquitination, and glycosylation, have been detected in soluble and aggregated α-syn in vivo. These PTMs can have either positive or negative effects on α-syn aggregation and toxicity, which may play critical roles in PD pathogenesis. Herein, we review the advances in synthetic and semisynthetic chemistry to generate homogeneous α-syn variants with site-specific modifications. Using these modified α-syn, we gain insight into the consequences of PTMs on α-syn aggregation and other biophysical properties, which can help elucidate the role of PTMs in the pathogenesis of PD and develop potential therapies to PD.

Segmental 13 C-Labeling and Raman Microspectroscopy of α-Synuclein Amyloid Formation.

Mapping conformational changes of α-synuclein (α-syn) from soluble, unstructured monomers to ß-sheet- rich aggregates is crucial towards understanding amyloid formation. Raman microspectroscopy is now used to spatially resolve conformational heterogeneity of amyloid aggregates and monitor amyloid formation of segmentally 13 C-labeled α-syn in real-time. As the 13 C-isotope shifts the amide-I stretching frequency to lower energy, the ligated construct, 13 C1-8612 CS87C-140 -α-syn, exhibits two distinct bands allowing for simultaneous detection of secondary structural changes in N-terminal 1-86 and C-terminal 87-140 residues. The disordered-to-ß-sheet conformational change is first observed for the N-terminal followed by the C-terminal region. Finally, Raman spectroscopic changes occurred prior to Thioflavin T fluorescence enhancement, indicating that the amide-I band is a superior probe of amyloid formation.

Generation and Characterization of Stable α-Synuclein Oligomers.

Alpha-synuclein oligomers are linked to the pathogenesis of Parkinson's disease and related neurodegenerative diseases. In this chapter, we present a method to generate kinetically stable α-synuclein oligomers by the addition of reactive aldehydes, 4-hydroxy-2-nonenal, and 4-oxo-2-nonenal. We also describe biochemical and immunological techniques to characterize the generated oligomers.

Transient ß-hairpin formation in α-synuclein monomer revealed by coarse-grained molecular dynamics simulation.

Parkinson's disease, originating from the intrinsically disordered peptide α-synuclein, is a common neurodegenerative disorder that affects more than 5% of the population above age 85. It remains unclear how α-synuclein monomers undergo conformational changes leading to aggregation and formation of fibrils characteristic for the disease. In the present study, we perform molecular dynamics simulations (over 180 µs in aggregated time) using a hybrid-resolution model, Proteins with Atomic details in Coarse-grained Environment (PACE), to characterize in atomic detail structural ensembles of wild type and mutant monomeric α-synuclein in aqueous solution. The simulations reproduce structural properties of α-synuclein characterized in experiments, such as secondary structure content, long-range contacts, chemical shifts, and (3)J(HNHCα )-coupling constants. Most notably, the simulations reveal that a short fragment encompassing region 38-53, adjacent to the non-amyloid-ß component region, exhibits a high probability of forming a ß-hairpin; this fragment, when isolated from the remainder of α-synuclein, fluctuates frequently into its ß-hairpin conformation. Two disease-prone mutations, namely, A30P and A53T, significantly accelerate the formation of a ß-hairpin in the stated fragment. We conclude that the formation of a ß-hairpin in region 38-53 is a key event during α-synuclein aggregation. We predict further that the G47V mutation impedes the formation of a turn in the ß-hairpin and slows down ß-hairpin formation, thereby retarding α-synuclein aggregation.

Site-specific differences in proteasome-dependent degradation of monoubiquitinated α-synuclein.

The formation of toxic aggregates composed largely of the protein α-synuclein are a hallmark of Parkinson's disease. Evidence from both early-onset forms of the disease in humans and animal models has shown that the progression of the disease is correlated with the expression levels of α-synuclein, suggesting that cellular mechanisms that degrade excess α-synuclein are key. We and others have shown that monoubiquitinated α-synuclein can be degraded by the 26S proteasome; however, the contributions of each of the nine known individual monoubiquitination sites were unknown. Herein, we determined the consequences of each of the modification sites using homogenous, semisynthetic proteins in combination with an in vitro proteasome turnover assay. The data suggest that the site-specific effects of monoubiquitination support different levels of α-synuclein degradation.

Chemical synthesis of proteins using peptide hydrazides as thioester surrogates.

This protocol provides a detailed procedure for the chemical synthesis of proteins through native chemical ligation of peptide hydrazides. The two crucial stages of this protocol are (i) the solid-phase synthesis of peptide hydrazides via Fmoc chemistry and (ii) the native chemical ligation of peptide hydrazides through in situ NaNO2 activation and thiolysis. This protocol may be of help in the synthesis of proteins that are not easily produced by recombinant technology and that include acid-sensitive modifications; it also does not involve the use of hazardous HF. The utility of the protocol is shown for the total synthesis of 140-aa-long α-synuclein, a protein that has an important role in the development of Parkinson's disease. The whole synthesis of the target protein α-synuclein in milligram scale takes ~30 working days.

Synthetic polyubiquitinated α-Synuclein reveals important insights into the roles of the ubiquitin chain in regulating its pathophysiology.

Ubiquitination regulates, via different modes of modifications, a variety of biological processes, and aberrations in the process have been implicated in the pathogenesis of several neurodegenerative diseases. However, our ability to dissect the pathophysiological relevance of the ubiquitination code has been hampered due to the lack of methods that allow site-specific introduction of ubiquitin (Ub) chains to a specific substrate. Here, we describe chemical and semisynthetic strategies for site-specific incorporation of K48-linked di- or tetra-Ub chains onto the side chain of Lys12 of α-Synuclein (α-Syn). These advances provided unique opportunities to elucidate the role of ubiquitination and Ub chain length in regulating α-Syn stability, aggregation, phosphorylation, and clearance. In addition, we investigated the cross-talk between phosphorylation and ubiquitination, the two most common α-Syn pathological modifications identified within Lewy bodies and Parkinson disease. Our results suggest that α-Syn functions under complex regulatory mechanisms involving cross-talk among different posttranslational modifications.

One-pot total chemical synthesis of human α-synuclein.

Post-translational modifications (PTMs) regulate key aspects of the physiological and pathogenic properties of Parkinson's disease-associated presynaptic protein α-synuclein. We herein describe a one-pot total chemical synthesis that should enable site-specific introduction of single or multiple PTMs or small molecule probes essentially at any site within the protein.

The clustering and spatial arrangement of beta-sheet sequence, but not order, govern alpha-synuclein fibrillogenesis.

The intrinsically unstructured protein alpha-synuclein (aS) is prone to misfold into cytotoxic beta-sheet-rich oligomers and amyloid fibrils that underlie the pathogenesis of Lewy body diseases such as Parkinson's disease. An important, recognized fibrillogenesis parameter is amino acid content, whereas the influence of amino acid sequence distribution is not as well understood. The fibril core of aS encompasses five regions of high beta-sheet propensity, termed beta1-beta5. Using four aS variants with identical amino acid compositions but rearranged pseudorepeat motifs, we show that beta2-beta5 sequence clustering, but not order, is important for efficient fibrillogenesis. For molecular species progressing toward the fibrillar state, order invariably increases; i.e., the spatial arrangement of sequence elements becomes restricted. By introducing disulfide bonds in a fibril structure-based manner, we demonstrated that a successful protofibril-to-fibril conversion is dependent upon the spatial arrangement of sequence elements of high beta-sheet propensity. Moreover, a disulfide-linked aS dimer is shown to fibrillize rapidly. We propose that a conformational search underlies the emergence of a fibrillar aS nucleus that is directed by gaps in sequence between beta-sheet regions and the accessible range of spatial beta-sheet arrangements in soluble, prefibrillar oligomers. On the basis of the universal cross-beta-sheet structure of amyloid fibrils, these principles are expected to apply to a wide range of amyloidogenic proteins.

Metal binding to alpha-synuclein peptides and its contribution to toxicity.

Recent studies have suggested that alpha-synuclein (AS) is a metal binding protein. Metals also induce protein aggregation. In order to clarify controversy over the location of the metal binding sites six peptide fragments spanning the full length of the protein were analysed to identify metal binding domains. Our results indicated that both the C-terminus of the protein and a region around histidine 50 play a role in copper binding. We suggest that the true binding domain is a nonlinear site composed of both areas acting together to bind copper. The toxicity of these peptides to SH-SY5Y cells was also studied. There was a copper-independent component associated with the NAC domain of the protein and a copper-dependent component associated with the C-terminus of the protein and potentiated by involvement of the N-terminus. We hypothesise that copper binding can cause conversion of AS to a neurotoxic form via inter-protein interactions.