Α-syn Structure Research Articles

An In-Silico Approach to Study the Effect of Phosphorylation (Ps129) on the Conformational Dynamics of Membrane Bound Α-Synuclein

Background: Phosphorylated α-Synuclein (α-Syn) is present in relatively small levels in normal human brains, but nearly all of the α-Syn in the Lewy bodies (LBs) that collect in the nigrostriatal region in the brain of Parkinson's disease patients is phosphorylated on serine 129 (pS129). Earlier studies suggested that mimicking phosphorylation at S129 may have an inhibitory effect on α-Syn aggregation and thus control α-Syn neuropathology. Although phosphorylation at S129 is associated with α-Syn inclusion in synucleinopathies, the mechanisms by which this post-translational modification (PTM) influences aggregation and contributes to LB illness in the brain are yet to be understood. Objective: This research aims to study the effect of phosphorylation (pS129) on the conformational dynamics of membrane-bound α-Syn using Molecular Dynamics (MD) simulations. Method: Using MD simulations, this computational study has demonstrated the effect of PTM on the conformational dynamics of pS129 α-Syn and its lipid membrane association. To better understand the impact of pS129 on the aggregation of the α-Syn structure monomer with recent atomic details, we have examined the MD trajectories, conducted a salt-bridge interaction study, Principal Component Analysis (PCA), and intra and inter-molecular hydrogen bond analysis. Results: The conformational structure of pS129 α-Syn was observed from the MD trajectory analysis to be stable throughout the simulation, with higher compactness and reduced flexibility. The stability of the structure of pS129 α-Syn was also evaluated by 2-D and 3-D principal component analysis followed by a free energy landscape plot showing the global minima. The conformational snapshots and Ramachandran plot showed the absence of α-strands in the α-Syn's Non-Amyloid Component Region (NAC) (71–82), which is necessary for aggregate formation. Conclusion: Further, the intermolecular hydrogen bonds analysis indicates that the NAC region is not embedded into the lipid bilayer and has limited association with the other regions of the protein. Our findings reveal salient features of pS129 modifications that inhibit α-Syn aggregation.

Computational investigation on the effect of the peptidomimetic inhibitors (NPT100-18A and NPT200-11) on the α-synuclein and lipid membrane interactions

Parkinson’s disease (PD) is associated with α-synuclein (α-Syn), a presynaptic protein that binds to cell membranes. The molecular pathophysiology of PD most likely begins with the binding of α-Syn to membranes. Recently, two peptidomimetic inhibitors (NPT100-18A and NPT200-11) were identified to potentially interact with α-Syn and affect the interaction of α-Syn with the membrane. In this study, the effect of the two peptidomimetic inhibitors on the α-Syn-membrane interaction was demonstrated. DFT calculations were performed for optimization of the two inhibitors, and the nucleophilicity (N) and electrophilicity (ω) of NPT100-18A and NPT200-11 were calculated to be 3.90 and 3.86 (N); 1.06 and 1.04 (ω), respectively. Using the docking tool (CB-dock2), the two α-Syn-peptidomimetic inhibitor complexes (α-Syn-NPT100-18A and α-Syn-NPT200-11) have been prepared. Then all-atom molecular dynamics (MD) simulation was carried out on the α-Syn (control), α-Syn-NPT100-18A and α-Syn-NPT200-11 complex systems in presence of DOPE: DOPS: DOPC (5:3:2) lipid bilayer. From the conformational dynamics analysis, the 3-D structure of α-Syn was found to be stable, and the helices present in the regions (1–37) and (45–95) of α-Syn were found to be retained in the presence of the two peptidomimetic inhibitors. The electron density profile analysis revealed the binding modes of NAC and C-terminal region of α-Syn (in the presence of NPT200-11 inhibitor) with lipid membrane are in the close vicinity from the lipid bilayer centre. Our findings in this study on α-Syn-membrane interactions may be useful for developing a new therapeutic approach for treating PD and other neurodegenerative disorders. Communicated by Ramaswamy H. Sarma

In Silico Evaluation of the Potential Association of the Pathogenic Mutations of Alpha Synuclein Protein with Induction of Synucleinopathies.

Alpha synuclein (α-Syn) is a neuronal protein encoded by the SNCA gene and is involved in the development of Parkinson's disease (PD). The objective of this study was to examine in silico the functional implications of non-synonymous single nucleotide polymorphisms (nsSNPs) in the SNCA gene. We used a range of computational algorithms such as sequence conservation, structural analysis, physicochemical properties, and machine learning. The sequence of the SNCA gene was analyzed, resulting in the mapping of 42,272 SNPs that are classified into different functional categories. A total of 177 nsSNPs were identified within the coding region; there were 20 variants that may influence the α-Syn protein structure and function. This identification was made by employing different analytical tools including SIFT, PolyPhen2, Mut-pred, SNAP2, PANTHER, PhD-SNP, SNP&Go, MUpro, Cosurf, I-Mut, and HOPE. Three mutations, V82A, K80E, and E46K, were selected for further examinations due to their spatial positioning within the α-Syn as determined by PyMol. Results indicated that these mutations may affect the stability and function of α-Syn. Then, a molecular dynamics simulation was conducted for the SNCA wildtype and the four mutant variants (p.A18G, p.V82A, p.K80E, and p.E46K). The simulation examined temperature, pressure, density, root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), solvent-accessible surface area (SASA), and radius of gyration (Rg). The data indicate that the mutations p.V82A, p.K80E, and p.E46K reduce the stability and functionality of α-Syn. These findings highlight the importance of understanding the impact of nsSNPs on α-syn structure and function. Our results required verifications in further protein functional and case-control studies. After being verified these findings can be used in genetic testing for the early diagnosis of PD, the evaluation of the risk factors, and therapeutic approaches.

Conformational dynamics of A30G α-synuclein that causes familial Parkinson disease

The first gene shown to be responsible for autosomal-dominant Parkinson’s disease (PD) is the SNCA gene, which encodes for alpha synuclein (α-Syn). Recently, a novel heterozygous A30G mutation of the SNCA gene associated with familial PD has been reported. However, little research has been done on how the A30G mutation affects the structure of α-Syn. So, using atomistic molecular dynamics (MD) simulation, we demonstrate here the key structural characteristics of A30G α-Syn in the free monomer form and membrane associated state. From the MD trajectory analysis, the structure of A30G α-Syn was noticed to exhibit rapid conformational change, increase in backbone flexibility near the site of mutation and decrease in α-helical propensity. The typical torsion angles in residues (Val26 and Glu28) near the mutation site were observed to deviate significantly in A30G α-Syn. In the case of membrane bound A30G α-Syn, the regions that were submerged in the lipid bilayer (N-helix (3-37) and turn region (38-44)) found to contain higher helical content than the elevated region above the lipid surface. The bending angle in the helix-N and helix-C regions were noticed to be relatively higher in the free form of A30G α-Syn (38.50) than in the membrane bound form (370). The A30G mutation in α-Syn was predicted to have an impact on the stability and function of the protein based on ΔΔG values obtained from the online servers. Our results demonstrate that the A30G mutation in α-Syn altered the protein’s α-helical structure and slightly altered the membrane binding. Communicated by Ramaswamy H. Sarma

Computer aided therapeutic tripeptide design, in alleviating the pathogenic proclivities of nocuous α-synuclein fibrils

Parkinson’s disorder (PD) exacerbates neuronal degeneration of motor nerves, thereby effectuating uncoordinated movements and tremors. Aberrant alpha-synuclein (α-syn) is culpable of triggering PD, wherein cytotoxic amyloid aggregates of α-syn get deposited in motor neurons to instigate neuro-degeneration. Amyloid aggregates, typically rich in beta sheets are cardinal targets to mitigate their neurotoxic effects. In this analysis, owing to their interaction specificity, we formulated an efficacious tripeptide out of the aggregation-prone region of α-syn protein. With the help of a proficient computational pipeline, systematic peptide shortening and an adept molecular simulation platform, we formulated a tripeptide, VAV from α-syn structure based hexapeptide KISVRV. Indeed, the VAV tripeptide was able to effectively mitigate the α-syn amyloid fibrils’ dynamic rate of beta-sheet formation. Additional trajectory analyses of the VAV- α-syn complex indicated that, upon its dynamic interaction, VAV efficiently altered the distinct pathogenic structural dynamics of α-syn, further advocating its potential in alleviating aberrant α-syn’s amyloidogenic proclivities. Consistent findings from various computational analyses have led us to surmise that VAV could potentially re-alter the pathogenic conformational orientation of α-syn, essential to mitigate its cytotoxicity. Hence, VAV tripeptide could be an efficacious therapeutic candidate to efficiently ameliorate aberrant α-syn amyloid mediated neurotoxicity, eventually attenuating the nocuous effects of PD. Communicated by Ramaswamy H. Sarma

Phosphatidylcholine and Phosphatidylserine Uniquely Modify the Secondary Structure of α-Synuclein Oligomers Formed in Their Presence at the Early Stages of Protein Aggregation.

Abrupt aggregation of α-synuclein (α-Syn) leads to a formation of highly toxic protein oligomers. These aggregates are the underlying molecular cause of an onset of the irreversible degeneration of dopaminergic neurons in midbrain, hypothalamus, and thalamus, a pathology known as Parkinson's disease. The transient nature of oligomers, as well as their structural and morphological heterogeneity, limits the use of cryo-electron microscopy and solid-state NMR, classical tools of structural biology, for elucidation of their secondary structure. Despite this limitation, numerous pieces of experimental evidence suggest that phospholipids can uniquely alter the structure and toxicity of oligomers. In this study, we utilize an innovative nano-infrared imaging technique, also known as atomic force microscopy infrared (AFM-IR) spectroscopy, to examine the structure of individual α-Syn oligomers grown in the presence of phosphatidylcholine (α-Syn:PC) and phosphatidylserine (α-Syn:PS). We determined the amount of the parallel and the antiparallel β-sheets, as well as the amount the α-helix and the unordered protein, in the secondary structure of α-Syn:PC and α-Syn:PS formed at day 2 (D2), 8 (D8), and 15 (D15) after initiation of protein aggregation. We found a gradual decrease in the amount of the parallel β-sheet in both α-Syn:PC and α-Syn:PS from D2 to D15 together with an increase in the α-helix and the unordered protein secondary structure. We infer that this is due to the presence of lipids in the structure of oligomers that prevent an expansion of the parallel β-sheet upon interaction of the oligomers with monomeric α-Syn.

Structural Basis for Dityrosine-Mediated Inhibition of α-Synuclein Fibrillization.

α-Synuclein (α-Syn) is an intrinsically disordered protein which self-assembles into highly organized β-sheet structures that accumulate in plaques in brains of Parkinson’s disease patients. Oxidative stress influences α-Syn structure and self-assembly; however, the basis for this remains unclear. Here we characterize the chemical and physical effects of mild oxidation on monomeric α-Syn and its aggregation. Using a combination of biophysical methods, small-angle X-ray scattering, and native ion mobility mass spectrometry, we find that oxidation leads to formation of intramolecular dityrosine cross-linkages and a compaction of the α-Syn monomer by a factor of √2. Oxidation-induced compaction is shown to inhibit ordered self-assembly and amyloid formation by steric hindrance, suggesting an important role of mild oxidation in preventing amyloid formation.

Beyond neurodegenerative diseases: α-synuclein in erythropoiesis

ABSTRACT α-synuclein (α-syn) is a highly conserved and thermostable protein that is widely distributed in human brain. An intracellular aggregation of α-syn in dopaminergic neurons is the hallmark of a group of neurodegenerative diseases including Parkinson’s disease. Interestingly, α-syn is also highly expressed in red blood cells and is considered as one of the most abundant proteins in red blood cells. Moreover, α-syn is thought to play a regulatory role during normal erythropoiesis. However, whether α-syn participates in the pathogenesis of erythroid diseases has not been reported. In this review, we discuss the protein structure of α-syn and the importance of α-syn in erythropoiesis.

Alpha-Synuclein PET Tracer Development-An Overview about Current Efforts.

Neurodegenerative diseases such as Parkinson’s disease (PD) are manifested by inclusion bodies of alpha-synuclein (α-syn) also called α-synucleinopathies. Detection of these inclusions is thus far only possible by histological examination of postmortem brain tissue. The possibility of non-invasively detecting α-syn will therefore provide valuable insights into the disease progression of α-synucleinopathies. In particular, α-syn imaging can quantify changes in monomeric, oligomeric, and fibrillic α-syn over time and improve early diagnosis of various α-synucleinopathies or monitor treatment progress. Positron emission tomography (PET) is a non-invasive in vivo imaging technique that can quantify target expression and drug occupancies when a suitable tracer exists. As such, novel α-syn PET tracers are highly sought after. The development of an α-syn PET tracer faces several challenges. For example, the low abundance of α-syn within the brain necessitates the development of a high-affinity ligand. Moreover, α-syn depositions are, in contrast to amyloid proteins, predominantly localized intracellularly, limiting their accessibility. Furthermore, another challenge is the ligand selectivity over structurally similar amyloids such as amyloid-beta or tau, which are often co-localized with α-syn pathology. The lack of a defined crystal structure of α-syn has also hindered rational drug and tracer design efforts. Our objective for this review is to provide a comprehensive overview of current efforts in the development of selective α-syn PET tracers.

Pharmacophore modeling and 3D-QSAR study for the design of novel α-synuclein aggregation inhibitors

Alpha-synuclein (α-syn), as a highly soluble presynaptic protein expressed in the brain, plays an important role in recycling synaptic vesicles and regulating the synthesis, storage, and release of neurotransmitters. Accumulation of α-syn in Lewy bodies and Lewy neurites is the pathological hallmark of Parkinson's disease (PD), so inhibition of α-syn aggregation may provide a novel approach for treating PD. In this study, the 3D structure of α-syn was downloaded from Protein Data Bank (PDB ID: 2N0A). A ligand-based pharmacophore model was conducted on a set of 43 diverse α-syn ligands, and the results suggested that two hydrogen-bond acceptors, one hydrophobic group, and two aromatic rings were significant to the inhibition of α-syn aggregation. A ligand-based 3D-QSAR model was also established with good statistical significance (R2 = 0.920) and excellent predictive ability (Q2 = 0.752). Novel indolinone derivatives were designed and synthesized based on the pharmacophore model. Subsequently, the 3D-QSAR model was used to predict the inhibitory activities towards α-syn aggregation, and the actual inhibitory activities were evaluated by thioflavin-T assay in vitro with the best inhibitory activity reaching 45.08%. The fitting results indicated that the built pharmacophore and 3D-QSAR models provided better reliability and accuracy for compound modification and prediction of the activity thereof. A ligand-based pharmacophore modeling and 3D-QSAR study have been performed on a set of 43 diverse ligands for α-synuclein for the first time. Based on the best pharmacophore modeling, novel indolinone derivatives were designed and synthesized, and the inhibitory activities for α-synuclein aggregation were evaluated by thioflavin-T assay in vitro, which preliminary indicated that five pharmacophore sites (two hydrogen bond acceptors (A), a hydrophobic group (H), and two aromatic rings (R)) in compounds contribute to the inhibitory activities. In the study, the built pharmacophore modeling and 3D-QSAR provided better reliability and accuracy for compound modification and prediction of the activity thereof.

α-Synuclein: An All-Inclusive Trip Around its Structure, Influencing Factors and Applied Techniques.

Alpha-synuclein (αSyn) is a highly expressed and conserved protein, typically found in the presynaptic terminals of neurons. The misfolding and aggregation of αSyn into amyloid fibrils is a pathogenic hallmark of several neurodegenerative diseases called synucleinopathies, such as Parkinson’s disease. Since αSyn is an Intrinsically Disordered Protein, the characterization of its structure remains very challenging. Moreover, the mechanisms by which the structural conversion of monomeric αSyn into oligomers and finally into fibrils takes place is still far to be completely understood. Over the years, various studies have provided insights into the possible pathways that αSyn could follow to misfold and acquire oligomeric and fibrillar forms. In addition, it has been observed that αSyn structure can be influenced by different parameters, such as mutations in its sequence, the biological environment (e.g., lipids, endogenous small molecules and proteins), the interaction with exogenous compounds (e.g., drugs, diet components, heavy metals). Herein, we review the structural features of αSyn (wild-type and disease-mutated) that have been elucidated up to present by both experimental and computational techniques in different environmental and biological conditions. We believe that this gathering of current knowledge will further facilitate studies on αSyn, helping the planning of future experiments on the interactions of this protein with targeting molecules especially taking into consideration the environmental conditions.

A Novel SNCA A30G Mutation Causes Familial Parkinsonʼs Disease

The SNCA gene encoding α-synuclein (αSyn) is the first gene identified to cause autosomal-dominant Parkinson's disease (PD). We report the identification of a novel heterozygous A30G mutation of the SNCA gene in familial PD and describe clinical features of affected patients, genetic findings, and functional consequences. Whole exome sequencing was performed in the discovery family proband. Restriction digestion with Bbvl was used to screen SNCA A30G in two validation cohorts. The Greek cohort included 177 familial PD probands, 109 sporadic PD cases, and 377 neurologically healthy controls. The German cohort included 136 familial PD probands, 380 sporadic PD cases, and 116 neurologically healthy controls. We also conducted haplotype analysis using 13 common single nucleotide variants around A30G to determine the possibility of a founder effect for A30G. We then used biophysical methods to characterize A30G αSyn. We identified a novel SNCA A30G (GRCh37, Chr4:90756730, c.89 C>G) mutation that co-segregated with the disease in five affected individuals of three Greek families and was absent from controls. A founder effect was strongly suggested by haplotype analysis. The A30G mutation had a local effect on the intrinsically disordered structure of αSyn, slightly perturbed membrane binding, and promoted fibril formation. Based on the identification of A30G co-segregating with the disease in three families, the absence of the mutation in controls and population databases, and the observed functional effects, we propose SNCA A30G as a novel causative mutation for familial PD. © 2021 International Parkinson and Movement Disorder Society.

Expression, purification and characterization of α-synuclein fibrillar specific scFv from inclusion bodies.

Aggregation of α-synuclein (α-syn) has been implicated in multiple neurodegenerative disorders including Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), collectively grouped as synucleinopathies. Recently, recombinant antibody fragments (Fab, scFvs and diabodies) against α-syn have emerged as an alternative to the traditional full-length antibody in immunotherapeutic approaches owing to their advantages including smaller size and higher stability, specificity and affinity. However, most of the recombinant antibody fragments tend to be expressed as inclusion bodies (IBs) making its purification extremely challenging. In the current study, a single-chain variable fragment (scFv-F) antibody, targeting the pathogenic α-syn fibrils, was engineered and expressed in E. coli. Majority of the expressed scFv-F accumulated in insoluble aggregates as IBs. A variety of mild and harsh solubilizing conditions were tested to solubilize IBs containing scFv-F to obtain the active protein. To preserve secondary structure and bioactivity, a mild solubilizing protocol involving 100 mM Tris, pH 12.5 with 2 M urea was chosen to dissolve IBs. Slow on-column refolding method was employed to subsequently remove urea and obtain active scFv-F. A three-dimensional (3D) model was built using homology modeling and subjected to molecular docking with the known α-syn structure. Structural alignment was performed to delineate the potential binding pocket. The scFv-F thus purified demonstrated high specificity towards α-syn fibrils compared to monomers. Molecular modeling studies suggest that scFv-F shares the same structural topology with other known scFvs. We present evidence through structural docking and alignment that scFv-F binds to α-syn C-terminal region. In conclusion, mild solubilization followed by slow on-column refolding can be utilized as a generalized and efficient method for hard to purify disease relevant insoluble proteins and/or antibody molecules from IBs.

Formation of α-synuclein aggregates in aqueous ethylammonium nitrate solutions.

The effect of adding ethylammonium nitrate (EAN), which is an ionic liquid (IL), on the aggregate formation of α-synuclein (α-Syn) in aqueous solution has been investigated. FTIR and Raman spectroscopy were used to investigate changes in the secondary structure of α-Syn and in the states of water molecules and EAN. The results presented here show that the addition of EAN to α-Syn causes the formation of an intermolecular β-sheet structure in the following manner: native disordered state → polyproline II (PPII)-helix → intermolecular β-sheet (α-Syn amyloid-like aggregates: α-SynA). Although cations and anions of EAN play roles in masking the charged side chains and PPII-helix-forming ability involved in the formation of α-SynA, water molecules are not directly related to its formation. We conclude that EAN-induced α-Syn amyloid-like aggregates form at hydrophobic associations in the middle of the molecules after masking the charged side chains at the N- and C-terminals of α-Syn.

Navigating the dynamic landscape of alpha-synuclein morphology: a review of the physiologically relevant tetrameric conformation.

N-acetylated α-synuclein (αSyn) has long been established as an intrinsically disordered protein associated with a dysfunctional role in Parkinson’s disease. In recent years, a physiologically relevant, higher order conformation has been identified as a helical tetramer that is tailored by buried hydrophobic interactions and is distinctively aggregation resistant. The canonical mechanism by which the tetramer assembles remains elusive. As novel biochemical approaches, computational methods, pioneering purification platforms, and powerful imaging techniques continue to develop, puzzling information that once sparked debate as to the veracity of the tetramer has now shed light upon this new counterpart in αSyn neurobiology. Nuclear magnetic resonance and computational studies on multimeric αSyn structure have revealed that the protein folding propensity is controlled by small energy barriers that enable large scale reconfiguration. Alternatively, familial mutations ablate tetramerization and reconfigure polymorphic fibrillization. In this review, we will discuss the dynamic landscape of αSyn quaternary structure with a focus on the tetrameric conformation.

Cerium oxide NPs mitigate the amyloid formation of α-synuclein and associated cytotoxicity.

AimAmong therapeutic proposals for amyloid-associated disorders, special attention has been given to the exploitation of nanoparticles (NPs) as promising agents against aggregation.MethodsIn this paper, the inhibitory effect of cerium oxide (CeO2) NPs against α-synuclein (α-syn) amyloid formation was explored by different methods such as Thioflavin T (ThT) and 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescence spectroscopy, Congo red adsorption assay, circular dichroism (CD) spectroscopy, transmission electron microscopy (TEM), and bioinformatical approaches. Also, the cytotoxicity of α-syn amyloid either alone or with CeO2 NPs against neuron-like cells (SH-SY5Y) was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flow cytometry, and quantitative real-time polymerase chain reaction (Bax and Bcl-2 gene expression) assays.ResultsThT and ANS fluorescence assays indicated that CeO2 NPs inhibit the formation of aggregated species and hydrophobic patches of α-syn in amyloidogenic conditions, respectively. Congo red and CD assays demonstrated that CeO2 NPs reduce the formation of amyloid species and β-sheets structures of α-syn molecules, respectively. TEM investigation also confirmed that CeO2 NPs limited the formation of well-defined fibrillary structures of α-syn molecules. Molecular docking and dynamic studies revealed that CeO2 NPs could bind with different affinities to α-syn monomer and amyloid species and fibrillar structure of α-syn is disaggregated in the presence of CeO2 NPs. Moreover, cellular assays depicted that CeO2 NPs mitigate the cell mortality, apoptosis, and the ratio of Bax/Bcl-2 gene expression associated with α-syn amyloids.ConclusionIt may be concluded that CeO2 NPs can be used as therapeutic agents to reduce the aggregation of proteins and mitigate the occurrence of neurodegenerative diseases.

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.

Impact of N-Terminal Acetylation on α-Synuclein Amyloid Formation

The accumulation of α-synuclein (αSyn) as β-sheet-rich amyloid in Lewy bodies is one of the hallmarks of Parkinson's disease. Recent data have demonstrated that αSyn is constitutively N-terminally acetylated in mammalian cells, a co-translational modification that has been shown to increase α-helical propensity in the N-terminal region of the protein and modulate membrane-binding behavior. The results of in vitro experiments comparing the amyloid formation kinetics of acetylated (Ac-αSyn) and the more widely studied non-acetylated a-synuclein (NH3-αSyn) have been contradictory. However, these assays largely rely on the extrinsic dye thioflavin T (ThT), which displays a dramatic increase in quantum yield upon binding to the cross-β sheet structure in amyloids. We find that the ThT response is approximately tenfold lower in Ac-αSyn, and that Ac-αSyn aggregates more slowly than NH3-αSyn at physiological pH. Intrinsic fluorescence of Trp mutants confirms that ThT emission accurately reports on the kinetics of amyloid formation for both forms of the protein. Seeding experiments indicate that the ThT fluorescence intensity is determined by the final fibrillar structure templated from the seeds rather than the presence or absence of the N-terminal acetyl group. Structural differences of the de novo and seeded fibrils are corroborated by Raman spectroscopy, limited proteolysis experiments, and transmission electron microscopy. These results indicate that N-terminal acetylation has important consequences for the aggregation propensity and amyloid structure of αSyn, and that nonacetylated αSyn is not a substitute for the biological form of the protein.

Amyloid fibril structure of α-synuclein determined by cryo-electron microscopy.

α-Synuclein (α-syn) amyloid fibrils are the major component of Lewy bodies, which are the pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. High-resolution structure of α-syn fibril is important for understanding its assembly and pathological mechanism. Here, we determined a fibril structure of full-length α-syn (1-140) at the resolution of 3.07 Å by cryo-electron microscopy (cryo-EM). The fibrils are cytotoxic, and transmissible to induce endogenous α-syn aggregation in primary neurons. Based on the reconstructed cryo-EM density map, we were able to unambiguously build the fibril structure comprising residues 37-99. The α-syn amyloid fibril structure shows two protofilaments intertwining along an approximate 21 screw axis into a left-handed helix. Each protofilament features a Greek key-like topology. Remarkably, five out of the six early-onset PD familial mutations are located at the dimer interface of the fibril (H50Q, G51D, and A53T/E) or involved in the stabilization of the protofilament (E46K). Furthermore, these PD mutations lead to the formation of fibrils with polymorphic structures distinct from that of the wild-type. Our study provides molecular insight into the fibrillar assembly of α-syn at the atomic level and sheds light on the molecular pathogenesis caused by familial PD mutations of α-syn.

Cardiolipin exposure on the outer mitochondrial membrane modulates α‐synuclein proteostasis in hPSC‐derived Parkinson's Disease neurons

IntroductionThe decline of voluntary motor function in Parkinson's disease (PD) is caused by loss of A9 dopaminergic (DA) neurons in the substantia nigra pars compacta. The death of these neurons is preceded by the formation of intracellular proteinaceous aggregates known as Lewy Bodies, which contain a variety of misfolded protein components, including α‐synuclein (α‐syn). Although SNCA mutations (α‐syn gene) are causal in several rare familial forms of PD, mutations in the SNCA locus are also consistently identified in genome‐wide association studies (GWAS) of sporadic PD, and α‐syn protein is commonly found in Lewy‐neurites of idiopathic disease cases. These findings emphasize the need to understand how altered α‐syn structure and function relate to PD pathogenesis in general terms.ApproachThe generation of human isogenic induced pluripotent stem cell (hiPSC) and embryonic stem cell (hESC) models of familial PD has facilitated the analysis of PD pathology at the cellular level, allowing us to contrast A9‐type DA neurons (hNs) harboring the SNCA‐A53T or SNCA‐E46K mutations with isogenic, mutation‐corrected controls. Using these systems in combination with SNCA‐A53T transgenic animals, we describe a novel mechanism in which the mitochondrial membrane lipid cardiolipin plays a vital role in preventing accumulation of α‐syn protein aggregates. This protective process is disrupted by both SNCA‐A53T and SNCA‐E46K mutations.ResultsHere, we demonstrate that cardiolipin translocates to the outer mitochondrial membrane (OMM) in both A53T and E46K SNCA‐mutant hNs at the onset of α‐syn deposition, where it binds α‐syn, facilitating the folding of intrinsically disordered α‐syn to an a‐helical conformation. This finding was confirmed in SNCA‐A53T transgenic mouse brains. Furthermore, we show that OMM‐localized cardiolipin can pull α‐syn monomers away from multimeric‐fibrils and facilitate their re‐folding, from multimeric b‐sheet forms back to monomers comprising a‐helices, effectively buffering α‐syn pathology. Surprisingly, both the A53T and E46K α‐syn mutations reduced the kinetic rate of cardiolipin‐mediated α‐syn re‐folding relative to wild‐type (WT), increasing the level of α‐syn present at the OMM in A53T and E46K hNs. Binding of A53T and E46K α‐syn to cardiolipin led to significantly increased LC3 recruitment to the OMM relative to WT α‐syn, which in turn increased mitochondrial stress, triggering excessive mitophagy.SignificanceThis work represents the first demonstration of α‐syn as a modulator of LC3 function, and identifies cardiolipin exposure on mitochondrial membranes as a key regulator of PD pathogenesis.Support or Funding InformationThis work was supported in part by the Parkinson Society of Canada (2014‐685 to SDR), the Natural Sciences and Engineering Research Council of Canada (RG060805 to SDR, RG121541 to GH), the CRC Program (GH).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.