Non-amyloid-β Component Research Articles

The Bifunctional Dimer Caffeine-Indan Attenuates α-Synuclein Misfolding, Neurodegeneration and Behavioral Deficits after Chronic Stimulation of Adenosine A1 Receptors.

We previously found that chronic adenosine A1 receptor stimulation with N6-Cyclopentyladenosine increased α-synuclein misfolding and neurodegeneration in a novel α-synucleinopathy model, a hallmark of Parkinson's disease. Here, we aimed to synthesize a dimer caffeine-indan linked by a 6-carbon chain to cross the blood-brain barrier and tested its ability to bind α-synuclein, reducing misfolding, behavioral abnormalities, and neurodegeneration in our rodent model. Behavioral tests and histological stains assessed neuroprotective effects of the dimer compound. A rapid synthesis of the 18F-labeled analogue enabled Positron Emission Tomography and Computed Tomography imaging for biodistribution measurement. Molecular docking analysis showed that the dimer binds to α-synuclein N- and C-termini and the non-amyloid-β-component (NAC) domain, similar to 1-aminoindan, and this binding promotes a neuroprotective α-synuclein "loop" conformation. The dimer also binds to the orthosteric binding site for adenosine within the adenosine A1 receptor. Immunohistochemistry and confocal imaging showed the dimer abolished α-synuclein upregulation and aggregation in the substantia nigra and hippocampus, and the dimer mitigated cognitive deficits, anxiety, despair, and motor abnormalities. The 18F-labeled dimer remained stable post-injection and distributed in various organs, notably in the brain, suggesting its potential as a Positron Emission Tomography tracer for α-synuclein and adenosine A1 receptor in Parkinson's disease therapy.

Understanding alpha-synuclein aggregation propensity in animals and humans

Alpha-synuclein (α-syn) aggregation plays a critical role in the pathogenicity of Parkinson's Disease (PD). This study aims to evaluate the aggregation propensity of α-syn fragment peptides designed using the variability found in humans and animals. Thioflavin T (ThT) and transmission electron microscopy (TEM) were used to validate the formation of fibrils to identify important amino acid residues. Human α-syn fragments 51–75, 37–61, 62–86, 76–100, and 116–140 demonstrate a significantly higher tendency to aggregate compared to fragments 1–25, 26–50, and 91–115. All species analyzed of the α-syn 37–61 and 62–86 regions were shown to form fibrils on both ThT and TEM. The α-syn 37–61 and 62–86 fragment regions exhibited a high susceptibility to aggregation, with fibril formation observed in all species. The A53T mutation in several α-syn 37–61 fragments may enhance their propensity for aggregation, suggesting a correlation between this mutation and the capacity for fibril formation. Furthermore, the presence of the non-amyloid-β component (NAC) region, specifically in α-syn 62–86, was consistently observed in several fragments that displayed fibril formation, indicating a potential correlation between the NAC region and the process of fibril formation in α-syn. Finally, the combination of a high quantity of valine and a low quantity of acidic amino acids in these fragments may serve as indicators of α-syn fibril formation.

Residues 2 to 7 of α-synuclein regulate amyloid formation via lipid-dependent and lipid-independent pathways

Amyloid formation by α-synuclein (αSyn) occurs in Parkinson's disease, multiple system atrophy, and dementia with Lewy bodies. Deciphering the residues that regulate αSyn amyloid fibril formation will not only provide mechanistic insight but may also reveal targets to prevent and treat disease. Previous investigations have identified several regions of αSyn to be important in the regulation of amyloid formation, including the non-amyloid-β component (NAC), P1 region (residues 36 to 42), and residues in the C-terminal domain. Recent studies have also indicated the importance of the N-terminal region of αSyn for both its physiological and pathological roles. Here, the role of residues 2 to 7 in the N-terminal region of αSyn is investigated in terms of their ability to regulate amyloid fibril formation in vitro and in vivo. Deletion of these residues (αSynΔN7) slows the rate of fibril formation in vitro and reduces the capacity of the protein to be recruited by wild-type (αSynWT) fibril seeds, despite cryo-EM showing a fibril structure consistent with those of full-length αSyn. Strikingly, fibril formation of αSynΔN7 is not induced by liposomes, despite the protein binding to liposomes with similar affinity to αSynWT. A Caenorhabditis elegans model also showed that αSynΔN7::YFP forms few puncta and lacks motility and lifespan defects typified by expression of αSynWT::YFP. Together, the results demonstrate the involvement of residues 2 to 7 of αSyn in amyloid formation, revealing a target for the design of amyloid inhibitors that may leave the functional role of the protein in membrane binding unperturbed.

In situ single-molecule investigations of the impacts of biochemical perturbations on conformational intermediates of monomeric α-synuclein.

α-Synuclein aggregation is a common trait in synucleinopathies, including Parkinson's disease. Being an unstructured protein, α-synuclein exists in several distinct conformational intermediates, contributing to both its function and pathogenesis. However, the regulation of these monomer conformations by biochemical factors and potential drugs has remained elusive. In this study, we devised an in situ single-molecule manipulation approach to pinpoint kinetically stable conformational intermediates of monomeric α-synuclein and explore the effects of various biochemical factors and drugs. We uncovered a partially folded conformation located in the non-amyloid-β component (NAC) region of monomeric α-synuclein, which is regulated by a preNAC region. This conformational intermediate is sensitive to biochemical perturbations and small-molecule drugs that influencing α-synuclein's aggregation tendency. Our findings reveal that this partially folded intermediate may play a role in α-synuclein aggregation, offering fresh perspectives for potential treatments aimed at the initial stage of higher-order α-synuclein aggregation. The single-molecule approach developed here can be broadly applied to the study of disease-related intrinsically disordered proteins.

Computational investigation on the conformational dynamics of C-terminal truncated α-synuclein bound to membrane

Accelerated progression rates in Parkinson’s disease (PD) have been linked to C-terminal domain (CTD) truncations of monomeric α-Synuclein (α-Syn), which have been suggested to increase amyloid aggregation in vivo and in vitro. In the brain of PD patients, CTD truncated α-Syn was found to have lower cell viability and tends to increase in the formation of fibrils. The CTD of α-Syn acts as a guard for regulating the normal functioning of α-Syn. The absence of the CTD may allow the N-terminal of α-Syn to interact with the membrane thereby affecting the normal functioning of α-Syn, and all of which will affect the etiology of PD. In this study, the conformational dynamics of CTD truncated α-Syn (1–99 and 1–108) monomers and their effect on the protein–membrane interactions were demonstrated using the all-atom molecular dynamics (MD) simulation method. From the MD analyses, it was noticed that among the two truncated monomers, α-Syn (1–108) was found to be more stable, shows rigidness at the N-terminal region and contains a significant number of intermolecular hydrogen bonds between the non-amyloid β-component (NAC) region and membrane, and lesser number of extended strands. Further, the bending angle in the N-terminal domain was found to be lesser in the α-Syn (1–108) in comparison with the α-Syn (1–99). Our findings suggest that the truncation on the CTD of α-Syn affects its interaction with the membrane and subsequently has an impact on the aggregation. Communicated by Ramaswamy H. Sarma

Exploration of Resveratrol as a Potent Modulator of α-Synuclein Fibril Formation.

The molecular determinants of amyloid protein misfolding and aggregation are key for the development of therapeutic interventions in neurodegenerative disease. Although small synthetic molecules, bifunctional molecules, and natural products offer a potentially advantageous approach to therapeutics to remodel aggregation, their evaluation requires new platforms that are informed at the molecular level. To that end, we chose pulsed hydrogen/deuterium exchange mass spectrometry (HDX-MS) to discern the phenomena of aggregation modulation for a model system of alpha synuclein (αS) and resveratrol, an antiamyloid compound. We invoked, as a complement to HDX, advanced kinetic modeling described here to illuminate the details of aggregation and to determine the number of oligomeric populations by kinetically fitting the experimental data under conditions of limited proteolysis. The misfolding of αS is most evident within and nearby the nonamyloid-β component region, and resveratrol significantly remodels that aggregation. HDX distinguishes readily a less solvent-accessible, more structured oligomer that coexists with a solvent-accessible, more disordered oligomer during aggregation. A view of the misfolding emerges from time-dependent changes in the fractional species across the protein with or without resveratrol, while details were determined through kinetic modeling of the protected species. A detailed picture of the inhibitory action of resveratrol with time and regional specificity emerges, a picture that can be obtained for other inhibitors and amyloid proteins. Moreover, the model reveals that new states of aggregation are sampled, providing new insights on amyloid formation. The findings were corroborated by circular dichroism and transmission electron microscopy.

Nucleation of α-Synuclein Amyloid Fibrils Induced by Cross-Interaction with β-Hairpin Peptides Derived from Immunoglobulin Light Chains.

Heterologous interactions between different amyloid-forming proteins, also called cross-interactions, may have a critical impact on disease-related amyloid formation. β-hairpin conformers of amyloid-forming proteins have been shown to affect homologous interactions in the amyloid self-assembly process. Here, we applied two β-hairpin-forming peptides derived from immunoglobulin light chains as models to test how heterologous β-hairpins modulate the fibril formation of Parkinson's disease-associated protein α-synuclein (αSyn). The peptides SMAhp and LENhp comprise β-strands C and C' of the κ4 antibodies SMA and LEN, which are associated with light chain amyloidosis and multiple myeloma, respectively. SMAhp and LENhp bind with high affinity to the β-hairpin-binding protein β-wrapin AS10 according to isothermal titration calorimetry and NMR spectroscopy. The addition of SMAhp and LENhp affects the kinetics of αSyn aggregation monitored by Thioflavin T (ThT) fluorescence, with the effect depending on assay conditions, salt concentration, and the applied β-hairpin peptide. In the absence of agitation, substoichiometric concentrations of the hairpin peptides strongly reduce the lag time of αSyn aggregation, suggesting that they support the nucleation of αSyn amyloid fibrils. The effect is also observed for the aggregation of αSyn fragments lacking the N-terminus or the C-terminus, indicating that the promotion of nucleation involves the interaction of hairpin peptides with the hydrophobic non-amyloid-β component (NAC) region.

Intramolecular interaction kinetically regulates fibril formation by human and mouse α-synuclein

Regulation of α-synuclein (αS) fibril formation is a potent therapeutic strategy for αS-related neurodegenerative disorders. αS, an intrinsically disordered 140-residue intraneural protein, comprises positively charged N-terminal, hydrophobic non-amyloid β component (NAC), and negatively charged C-terminal regions. Although mouse and human αS share 95% sequence identity, mouse αS forms amyloid fibrils faster than human αS. To evaluate the kinetic regulation of αS fibrillation, we examined the effects of mismatched residues in human and mouse αS on fibril formation and intramolecular interactions. Thioflavin T fluorescence assay using domain-swapped or C-terminal-truncated αS variants revealed that mouse αS exhibited higher nucleation and fibril elongation than human αS. In mouse αS, S87N substitution in the NAC region rather than A53T substitution is dominant for enhanced fibril formation. Fӧrester resonance energy transfer analysis demonstrated that the intramolecular interaction of the C-terminal region with the N-terminal and NAC regions observed in human αS is perturbed in mouse αS. In mouse αS, S87N substitution is responsible for the perturbed interaction. These results indicate that the interaction of the C-terminal region with the N-terminal and NAC regions suppresses αS fibril formation and that the human-to-mouse S87N substitution in the NAC region accelerates αS fibril formation by perturbing intramolecular interaction.

Unveiling How Hydroxytyrosol Destabilizes α-Syn Oligomers Using Molecular Simulations.

The etiology of Parkinson's disease (PD) is mainly linked to the α-synuclein (α-Syn) fibrillogenesis. Hydroxytyrosol (HT), also known as 3,4-dihydroxyphenylethanol, is a naturally occurring polyphenol, found in extra virgin olive oil, and has been shown to have cardioprotective, anticancer, antiobesity, and antidiabetic properties. HT has neuroprotective benefits in neurodegenerative diseases and lessens the severity of PD by reducing the aggregation of α-Syn and destabilizing the preformed toxic α-Syn oligomers. However, the molecular mechanism by which HT destabilizes α-Syn oligomers and alleviates the accompanying cytotoxicity remains unexplored. The impact of HT on the α-Syn oligomer structure and its potential binding mechanism was examined in this work by employing molecular dynamics (MD) simulations. The secondary structure analysis depicted that HT significantly reduces the β-sheet and concomitantly increases the coil content of α-Syn trimer. Visualization of representative conformations from the clustering analysis depicted the hydrogen bond interactions of the hydroxyl groups in HT with the N-terminal and nonamyloid-β component (NAC) region residues of α-Syn trimer, which, in turn, leads to the weakening of interchain interactions in α-Syn trimer and resulted in the disruption of the α-Syn oligomer. The binding free energy calculations depict that HT binds favorably to α-Syn trimer (ΔGbinding = -23.25 ± 7.86 kcal/mol) and a notable reduction in the interchain binding affinity of α-Syn trimer on the incorporation of HT, which, in turn, highlights its potential to disrupt α-Syn oligomers. The current research provided mechanistic insights into the destabilization of α-Syn trimer by HT, which, in turn, will provide new clues for developing therapeutics against PD.

Inhibitory mechanism of n-MTAB AuNPs for α-synuclein aggregation.

The aggregation of alpha-synuclein (α-syn) is closely related to the pathogenesis anddysfunction of Parkinson's disease. To investigate the potential of nanoparticlemediatedtherapy, the interactive mechanism between α-syn and n-myristyltrimethylammonium bromide (MTAB)Gold nanoparticles (AuNPs) with different diameters was explored by molecular dynamics simulations. The results indicated that there was a directional interaction between α-syn and n-MTABAuNPs, in which the driving force for the binding of the C-terminus in α-syn came from electrostatic interactions andthe nonamyloid β component (NAC) domain exhibited weak hydrophobic interactions as well as electrostaticinteraction, thereby preventing α-syn aggregation. Energy statistics and analysis showed that for 5-MTAB AuNPs,acidic amino acids such as Glu and Asp played a very important role. This studynot only demonstrated a theoretical foundation for the behavior of biomolecules directionally adsorbed on thesurface of biofunctional nanoparticles but also indicated that 5-MTAB AuNPs may be a potential inhibitor againstα-syn protein aggregation.

Aggregation of a Parkinson's Disease-Related Peptide: When Does Urea Weaken Hydrophobic Interactions?

While the exact cause of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease is not completely understood, compelling evidence implicates the aggregation of specific proteins and peptides. Co-solvents can provide molecular insight into protein aggregation mechanisms and the chemical nature of potential aggregation inhibitors. Here, we study, through molecular simulations, the hydration and binding free energies of an amphiphilic peptide from the nonamyloid-β component (NAC), a key aggregation-prone domain of α-synuclein, in water and an 8 M aqueous urea solution. Isoleucine, glycine, and serine peptides of the same length are also studied to unravel the role of urea in the hydration and aggregation of hydrophobic and hydrophilic domains. A strong impact of urea in hindering the aggregation of the NAC subdomain is observed. A slightly weaker aggregation inhibition is observed for the Gly and Ser peptides, whereas a much lower aggregation inhibitory activity is found for the Ile peptide, seemingly contrasting with urea's protein unfolding mechanism. This behavior is shown to derive from a lower profusion of urea next to the hydrophobic side chains and the backbone of the Ile's peptide in the dimeric form. As a consequence, β-sheets, formed upon aggregation, remain nearly intact. Hydrophilic neighbor groups in the amphiphilic NAC subdomain, however, are shown to anchor enough urea to weaken hydrophobic interactions and disrupt β-sheet structures. Our results indicate that urea's activity is potentiated in amphiphilic domains and that potential drugs could disrupt hydrophobic β-sheet-rich regions while not binding primarily to hydrophobic amino acids.

Kisspeptin-10 Rescues Cholinergic Differentiated SHSY-5Y Cells from α-Synuclein-Induced Toxicity In Vitro.

The neuropathological substrate of dementia with Lewy bodies (DLB) is defined by the inextricable cross-seeding accretion of amyloid-β (Aβ) and α-synuclein (α-syn)-laden deposits in cholinergic neurons. The recent revelation that neuropeptide kisspeptin-10 (KP-10) is able to mitigate Aβ toxicity via an extracellular binding mechanism may provide a new horizon for innovative drug design endeavors. Considering the sequence similarities between α-syn’s non-amyloid-β component (NAC) and Aβ’s C-terminus, we hypothesized that KP-10 would enhance cholinergic neuronal resistance against α-syn’s deleterious consequences through preferential binding. Here, human cholinergic SH-SY5Y cells were transiently transformed to upsurge the mRNA expression of α-syn while α-syn-mediated cholinergic toxicity was quantified utilizing a standardized viability-based assay. Remarkably, the E46K mutant α-syn displayed elevated α-syn mRNA levels, which subsequently induced more cellular toxicity compared with the wild-type α-syn in choline acetyltransferase (ChAT)-positive cholinergic neurons. Treatment with a high concentration of KP-10 (10 µM) further decreased cholinergic cell viability, while low concentrations of KP-10 (0.01–1 µM) substantially suppressed wild-type and E46K mutant α-syn-mediated toxicity. Correlating with the in vitro observations are approximations from in silico algorithms, which inferred that KP-10 binds favorably to the C-terminal residues of wild-type and E46K mutant α-syn with CDOCKER energy scores of −118.049 kcal/mol and −114.869 kcal/mol, respectively. Over the course of 50 ns simulation time, explicit-solvent molecular dynamics conjointly revealed that the docked complexes were relatively stable despite small-scale fluctuations upon assembly. Taken together, our findings insinuate that KP-10 may serve as a novel therapeutic scaffold with far-reaching implications for the conceptualization of α-syn-based treatments.

Crocin Inhibits the Fibrillation of Human α-synuclein and Disassembles Mature Fibrils: Experimental Findings and Mechanistic Insights from Molecular Dynamics Simulation.

The aggregation of human alpha-synuclein (hαS) is pivotally implicated in the development of most types of synucleinopathies. Molecules that can inhibit or reverse the aggregation process of amyloidogenic proteins have potential therapeutic value. The anti-aggregating activity of multiple carotenoid compounds has been reported over the past decades against a growing list of amyloidogenic polypeptides. Here, we aimed to determine whether crocin, the main carotenoid glycoside component of saffron, would inhibit hαS aggregation or could disassemble its preformed fibrils. By employing a series of biochemical and biophysical techniques, crocin was exhibited to inhibit hαS fibrillation in a dose-dependent fashion by stabilizing very early aggregation intermediates in off-pathway non-toxic conformations with little β-sheet content. We also observed that crocin at high concentrations could efficiently destabilize mature fibrils and disassemble them into seeding-incompetent intermediates by altering their β-sheet conformation and reshaping their structure. Our atomistic molecular dynamics (MD) simulations demonstrated that crocin molecules bind to both the non amyloid-β component (NAC) region and C-terminal domain of hαS. These interactions could thereby stabilize the autoinhibitory conformation of the protein and prevent it from adopting aggregation-prone structures. MD simulations further suggested that ligand molecules prefer to reside longitudinally along the fibril axis onto the edges of the inter-protofilament interface where they establish hydrogen and hydrophobic bonds with steric zipper stabilizing residues. These interactions turned out to destabilize hαS fibrils by altering the interstrand twist angles, increasing the rigidity of the fibril core, and elevating its radius of gyration. Our findings suggest the potential pharmaceutical implication of crocin in synucleinopathies.

Movement of prion-like α-synuclein along the gut-brain axis in Parkinson's disease: A potential target of curcumin treatment.

A pathological hallmark of the neurodegenerative disorder, Parkinson's disease (PD), is aggregation of toxic forms of the presynaptic protein, α-synuclein in structures known as Lewy bodies. α-Synuclein pathology is found in both the brain and gastrointestinal tracts of affected individuals, possibly due to the movement of this protein along the vagus nerve that connects the brain to the gut. In this review, we discuss current insights into the spread of α-synuclein pathology along the gut-brain axis, which could be targeted for therapeutic interventions. The prion-like propagation of α-synuclein, and the clinical manifestations of gastrointestinal dysfunction in individuals living with PD, are discussed. There is currently insufficient evidence that surgical alteration of the vagus nerve, or removal of gut-associated lymphoid tissues, such as the appendix and tonsils, are protective against PD. Furthermore, we propose curcumin as a potential candidate to prevent the spread of α-synuclein pathology in the body by curcumin binding to α-synuclein's non-amyloid β-component (NAC) domain. Curcumin is an active component of the food spice turmeric and is known for its antioxidant, anti-inflammatory, and potentially neuroprotective properties. We hypothesize that once α-synuclein is bound to curcumin, both molecules are subsequently excreted from the body. Therefore, dietary supplementation with curcumin over one's lifetime has potential as a novel approach to complement existing PD treatment and/or prevention strategies. Future studies are required to validate this hypothesis, but if successful, this could represent a significant step towards improved nutrient-based therapeutic interventions and preventative strategies for this debilitating and currently incurable disorder.

Potential Neuroprotection for Equine Pituitary Pars Intermedia Dysfunction

Cushing's disease, known as pituitary pars intermedia dysfunction (PPID), is the most commonly diagnosed equine endocrinopathy. Compelling evidence suggests that alpha-synuclein, an abundant neuronal protein, can acquire a neurotoxic feature that kills dopamine secreting nerve cells in horse afflicted with PPID. Horseswith PPID have higher levels ofalpha-synuclein in the dopaminergic nerve terminals withinthe pars intermedia of the pituitary gland compared withhealthy horses. Using protein extracts from pituitary glands of horses with PPID, biophysical assays, and bioinformatic analysis, we have identified that equine alpha-synuclein has high propensity to convert (misfold and aggregate) into this toxic form. Our drug discovery lab focusses on identifying and designing small molecules to impede the formation of intermediate species and fibrils. Over the last decades, natural products have emerged as neuroprotective agents for chronic neurodegenerative diseases. We hypothesize that a natural product such as resveratrol locks equine alpha-synuclein molecules into a conformation unable to form cytotoxic oligomers and fibrils, thereby conferring cytoprotection. Most recently, we have identified the region 62-86 in the equine alpha-synuclein that is highly prone to convert into a toxic conformation. Fragment 62-86 is part of the non-amyloid-β component (NAC), a hydrophobic region that may contribute to alpha-synuclein oligomerization. The carboxy-terminal region (90-140) exhibits the lowest aggregation score. This region is intrinsically disordered and may contribute minimally to the aggregation. We conducted a pilot screening with silibinin and resveratrol (both flavonoids and natural products) on the different equine and human alpha-synuclein fragment peptides with thioflavin T fluorescence (ThT) assay. Resveratrol decreased relative ThT fluorescence of equine alpha-synuclein fragment 62-86 by ~70%. Treating equine alpha-synuclein fragment 62-86 with resveratrol reduced the number of alpha-synuclein fibrils as assessed by transmission electron microscopy. This project will set the stage to move into a lead optimization program and underscore the value of equine alpha-synuclein in PPID drug discovery.

Membrane Interactions of α-Synuclein Revealed by Multiscale Molecular Dynamics Simulations, Markov State Models, and NMR.

α-Synuclein (αS) is a presynaptic protein that binds to cell membranes and is linked to Parkinson’s disease (PD). Binding of αS to membranes is a likely first step in the molecular pathophysiology of PD. The αS molecule can adopt multiple conformations, being largely disordered in water, adopting a β-sheet conformation when present in amyloid fibrils, and forming a dynamic multiplicity of α-helical conformations when bound to lipid bilayers and related membrane-mimetic surfaces. Multiscale molecular dynamics simulations in conjunction with nuclear magnetic resonance (NMR) and cross-linking mass spectrometry (XLMS) measurements are used to explore the interactions of αS with an anionic lipid bilayer. The simulations and NMR measurements together reveal a break in the helical structure of the central non-amyloid-β component (NAC) region of αS in the vicinity of residues 65–70, which may facilitate subsequent oligomer formation. Coarse-grained simulations of αS starting from the structure of αS when bound to a detergent micelle reveal the overall pattern of protein contacts to anionic lipid bilayers, while subsequent all-atom simulations provide details of conformational changes upon membrane binding. In particular, simulations and NMR data for liposome-bound αS indicate incipient β-strand formation in the NAC region, which is supported by intramolecular contacts seen via XLMS and simulations. Markov state models based on the all-atom simulations suggest a mechanism of conformational change of membrane-bound αS via a dynamic helix break in the region of residue 65 in the NAC region. The emergent dynamic model of membrane-interacting αS advances our understanding of the mechanism of PD, potentially aiding the design of novel therapeutic approaches.

Dimerization of α-Synuclein Fragments Studied by Isothermal-Isobaric Replica-Permutation Molecular Dynamics Simulation.

Aggregates and fibrils of intrinsically disordered α-synuclein are associated with Parkinson's disease. Within a non-amyloid β component (NAC) spanning from the 61st to the 95th residue of α-synuclein, an 11-residue segment called NACore (68GAVVTGVTAVA78) is an essential region for both fibril formation and cytotoxicity. Although NACore peptides alone are known to form aggregates and amyloid fibrils, the mechanisms of aggregation and fibrillation remain unknown. This study investigated the dimerization process of NACore peptides as the initial stage of the aggregation and fibrillation processes. We performed an isothermal-isobaric replica-permutation molecular dynamics simulation, which is one of the efficient sampling methods, for the two NACore peptides in explicit water over 96 μs. The simulation succeeded in sampling a variety of dimer structures. An analysis of secondary structure revealed that most of the NACore dimers form intermolecular β-bridges. In particular, more antiparallel β-bridges were observed than parallel β-bridges. We also found that intramolecular secondary structures such as α-helix and antiparallel β-bridge are stabilized in the pre-dimer state. However, we identified that the intermolecular β-bridges tend to form directly between residues with no specific structure rather than via the intramolecular β-bridges. This is because the NACore peptides still have a low propensity to form the intramolecular secondary structures even though they are stabilized in the pre-dimer state.

Similarity of the non-amyloid-β component and C-terminal tail of monomeric and tetrameric alpha-synuclein with 14-3-3 sigma

Alpha-synuclein (αSyn) is often described as a predominantly disordered protein that has a propensity to self-assemble into toxic oligomers that are found in patients with Parkinson's and Alzheimer's diseases. αSyn's chaperone behavior and tetrameric structure are proposed to be protective against toxic oligomerization. In this paper, we extended the previously proposed similarity between αSyn and 14-3-3 proteins to the α-helical tetrameric species of αSyn in detail. 14-3-3 proteins are a family of well-folded proteins with seven human isoforms, and function in signal transduction and as molecular chaperones. We investigated protein homology, using sequence alignment, amyloid, and disorder prediction, as well as three-dimensional visualization and protein-interaction networks. Our results show sequence homology and structural similarity between the aggregation-prone non-amyloid-β component (NAC) residues Val-52 to Gly-111 in αSyn and 14-3-3 sigma residues Leu-12 to Gly-78. We identified an additional region of sequence homology in the C-terminal region of αSyn (residues Ser-129 to Asp-135) and a C-terminal loop of 14-3-3 between helix αH and αI (residues Ser-209 to Asp-215). This data indicates αSyn shares conserved domain architecture with small heat shock proteins. We show predicted regions of high amyloidogenic propensity and intrinsic structural disorder in αSyn coincide with amyloidogenic and disordered predictions for 14-3-3 proteins. The homology in the NAC region aligns with residues involved in dimer- and tetramerization of the non-amyloidogenic 14-3-3 proteins. Because 14-3-3 proteins are generally not prone to misfolding, our results lend further support to the hypothesis that the NAC region is critical to the assembly of αSyn into the non-toxic tetrameric state.

Interaction of Alpha-Synuclein and Its Mutants with Rigid Lipid Vesicle Mimics of Varying Surface Curvature.

Abnormal aggregation of alpha-synuclein (α-syn), an intrinsically disordered neuronal protein, is strongly implicated in the development of Parkinson's disease. Efforts to better understand α-syn's native function and its pathogenic role in neurodegeneration have revealed that the protein interacts with anionic lipid vesicles via adoption of an amphipathic α-helical structure; however, the ability of α-syn to remodel lipid membranes has made it difficult to decipher the role of vesicle surface curvature in protein binding behavior. In this study, sodium dodecyl sulfate (SDS)-coated gold nanoparticles (AuNPs), which mimic bilayer vesicle architecture, were synthesized in order to conduct a systematic investigation into the binding interaction of α-syn and two of its mutants (A30P and E46K) with rigid lipid vesicle mimics of defined surface curvature. By incorporating a rigid AuNP core (∼10-100 nm), the ability of α-syn to remodel the vesicle mimics was removed and their surface curvature could be fixed. Proteomics studies showed that, upon binding of free α-syn to the surface of SDS-AuNPs, the N-terminus of α-syn became less solvent accessible, whereas its C-terminus became more accessible. Interestingly, α-syn's non-amyloid-β component (NAC) region also exhibited increased solvent accessibility, suggesting that α-syn bound to rigid vesicle-like structures could possess heightened aggregation propensity and therefore pathogenicity. Additionally, both the A30P and E46K mutations were found to adopt distinct binding modes on the mimics' surface. In contrast with previous reports, similar binding affinities were observed for WT, A30P, and E46K α-syn toward SDS-AuNPs of all sizes, indicating the potential importance of vesicle deformability in determining α-syn binding behavior.

A short motif in the N-terminal region of α-synuclein is critical for both aggregation and function.

Aggregation of human α-synuclein (αSyn) is linked to Parkinson’s disease (PD) pathology. The central region of the αSyn sequence contains the non-amyloid β-component (NAC) crucial for aggregation. However, how NAC flanking regions modulate αSyn aggregation remains unclear. Using bioinformatics, mutation, and NMR we identify a 7-residue sequence, named P1 (residues 36-42), that controls αSyn aggregation. Deletion or substitution of this ‘master-controller’ prevents aggregation at pH 7.5 in vitro. At lower pH, P1 synergises with a sequence containing the PreNAC region (P2, residues 45-57) to prevent aggregation. Deleting P1 (ΔP1) or both P1 and P2 (ΔΔ) also prevents age-dependent αSyn aggregation and toxicity in C. elegans models and prevents αSyn-mediated vesicle fusion by altering the conformational properties of the protein when lipid-bound. The results highlight the importance of a master-controller sequence motif that controls both αSyn aggregation and function- a region that could be targeted to prevent aggregation in disease.