α-Synuclein Drives Tau's Cytotoxic Aggregates Formation through Hydrophobic Interactions.

Tau and α-synuclein are proteins involved in pathologies known as tauopathies and synucleinopathies, respectively. Moreover, evidence shows that there is a crosstalk between them as is seen in the brains of individuals with sporadic neurodegenerative disorders. Based on that, we present data showing that the hydrophobic α-peptide 71 VTGVTAVAQKTV82 induces the aggregation of the full-length tau fragment in the absence of heparin assessed by ThT. Moreover, AFM images reveal the presence of straight filaments and amorphous aggregates of full-length tau in the presence of the α-peptide. Additionally, ITC experiments showed the interaction of the α-peptide with tau full-length (441 amino acids),4R (amino acids from 244 to 369), and both hexapeptides 275 VQIINK280 and 306 VQIVYK311 through hydrophobic interactions. The Raman spectroscopy spectra showed conformational changes in the Amide region in the aggregates formed with full-length tau and α-syn peptide. Furthermore, the incubation of extracellular aggregates with N2a cells showed morphological differences in the cellular body and the nucleus suggesting cell death. Moreover,, the incubation of different types of aggregates in cell culture provokes the release of Lactate dehydrogenase (LDH). Altogether, we found that α-synuclein peptide can drive the aggregation of full-length tau-provoking morphological and structural changes evoking cytotoxic effects.

Heterotypic liquid-liquid phase separation of tau and α-synuclein: Implications for overlapping neuropathologies.

Tauopathies and synucleinopathies are characterized by the aggregation of Tau and α-synuclein (AS) into amyloid structures, respectively. Individuals with these neuropathies have an elevated risk of developing subsequent neurodegenerative or comorbid disorders. Intriguingly, post-mortem brain examinations have revealed co-localization of Tau and AS aggregates, suggesting a synergistic pathological relationship with an adverse prognosis. The role of liquid-liquid phase separation (LLPS) in the development of neurodegenerative diseases is currently receiving significant attention, as it can contribute to the aggregation and co-deposition of amyloidogenic proteins. In this study, we investigated the phase separation behavior of Tau and AS under various insults, some of which are implicated in disease progression. Our findings demonstrate the formation of heterotypic droplets composed of Tau and AS at physiologically relevant mole ratios that mimic neurons' soma and terminal buttons. Importantly, these heterotypic droplets exhibit increased resistance to electrostatic screening compared to homotypic condensates. Moreover, we observed that biologically relevant biomolecules, known to be dysregulated in disease, exert different effects on these droplets. Additionally, we provide evidence that phase separation itself influences the amyloid aggregation of Tau and AS, underscoring the significance of this process in the development of aggregopathies.

How oxidized EGCG remodels α-synuclein fibrils into non-toxic aggregates: insights from computational simulations.

The misfolding and aggregation of the presynaptic protein α-synuclein (α-syn) is a pathological hallmark of Parkinson's disease (PD). Targeting α-syn has emerged as a promising therapeutic strategy for PD. Emerging in vitro evidence supports a dual action of epigallocatechin-3-gallate (EGCG) against amyloid neurotoxicity. EGCG can halt the formation of toxic aggregates by redirecting the amyloid fibril aggregation pathway toward non-toxic aggregates and remodeling the existing toxic fibrils into non-toxic aggregates. Moreover, EGCG oxidation can enhance fibril's remodeling by forming Schiff bases, leading to crosslinking of the fibril. However, this covalent modification is not required for amyloid remodeling, and establishing non-specific hydrophobic interactions with sidechains seems to be the main driver of amyloid remodeling by EGCG. Thioflavin (ThT) is a gold standard probe to detect amyloid fibrils in vitro, and oxidized EGCG competes with ThT for amyloid fibrils' binding sites. In this work, we performed docking and molecular dynamics (MD) simulations to gain insights into the intermolecular interactions of oxidized EGCG and ThT with a mature α-syn fibril. We find that oxidized EGCG moves within lysine-rich sites within the hydrophobic core of the α-syn fibril, forming aromatic and hydrogen-bonding (H-bond) interactions with different residues during the whole MD simulation time. In contrast, ThT, which does not remodel amyloid fibrils, was docked to the same sites but only via aromatic interactions. Our findings suggest that non-covalent interactions play a role in oxidized EGCG binding into the hydrophobic core, including H-bond and aromatic interactions with some residues in the amyloid remodeling processes. These interactions would ultimately lead to a disturbance of structural features as determinants for stabilizing this fibril into a compact and pathogenic Greek key topology.

Oxidative Stress and Mitochondrial Complex I Dysfunction Correlate with Neurodegeneration in an α-Synucleinopathy Animal Model.

The α-synucleinopathies constitute a subset of neurodegenerative disorders, of which Parkinson's disease (PD) is the most common worldwide, characterized by the accumulation of misfolded α-synuclein in the cytoplasm of neurons, which spreads in a prion-like manner to anatomically interconnected brain areas. However, it is not clear how α-synucleinopathy triggers neurodegeneration. We recently developed a rat model through a single intranigral administration of the neurotoxic ß-sitosterol ß-D-glucoside (BSSG), which produces α-synucleinopathy. In this model, we aimed to evaluate the temporal pattern of levels in oxidative and nitrosative stress and mitochondrial complex I (CI) dysfunction and how these biochemical parameters are associated with neurodegeneration in different brain areas with α-synucleinopathy (Substantia nigra pars compacta, the striatum, in the hippocampus and the olfactory bulb, where α-syn aggregation spreads). Interestingly, an increase in oxidative stress and mitochondrial CI dysfunction accompanied neurodegeneration in those brain regions. Furthermore, in silico analysis suggests a high-affinity binding site for BSSG with peroxisome proliferator-activated receptors (PPAR) alpha (PPAR-α) and gamma (PPAR-γ). These findings will contribute to elucidating the pathophysiological mechanisms associated with α-synucleinopathies and lead to the identification of new early biomarkers and therapeutic targets.

Aromaticity at position 39 in α-synuclein: A modulator of amyloid fibril assembly and membrane-bound conformations.

Recent studies revealed that molecular events related with the physiology and pathology of αS might be regulated by specific sequence motifs in the primary sequence of αS. The importance of individual residues in these motifs remains an important open avenue of investigation. In this work, we have addressed the structural details related to the amyloid fibril assembly and lipid-binding features of αS through the design of site-directed mutants at position 39 of the protein and their study by in vitro and in vivo assays. We demonstrated that aromaticity at position 39 of αS primary sequence influences strongly the aggregation properties and the membrane-bound conformations of the protein, molecular features that might have important repercussions for the function and dysfunction of αS. Considering that aggregation and membrane damage is an important driver of cellular toxicity in amyloid diseases, future work is needed to link our findings with studies based on toxicity and neuronal cell death. BRIEF STATEMENT OUTLINING SIGNIFICANCE: Modulation by distinct sequential motifs and specific residues of αS on its physiological and pathological states is an active area of research. Here, we demonstrated that aromaticity at position 39 of αS modulates the membrane-bound conformations of the protein, whereas removal of aromatic functionality at position 39 reduces strongly the amyloid assembly in vitro and in vivo. Our study provides new evidence for the modulation of molecular events related with the physiology and pathology of αS.

Comparing the copper binding features of alpha and beta synucleins.

Amyloid aggregation of α-synuclein (AS) is one of the hallmarks of Parkinson's disease (PD). Copper ions specifically bind at the N-terminus of AS, accelerating protein aggregation. Its protein homolog ß-synuclein (BS) is also a copper binding protein, but it inhibits AS aggregation. Here, a comparative spectroscopic study of the Cu2+ binding properties of AS and BS has been performed, using electronic absorption, circular dichroism (CD) and electronic paramagnetic resonance (EPR). Our comparative spectroscopic study reveals striking similarities between the Cu2+ binding features of the two proteins. The Cu2+ binding site at the N-terminal group of BS protein, modeled by the BS (1-15) fragment is identical to that of AS; however, its rate of reduction is three times faster as compared to the AS site, consistent with BS having an additional Met residue in its Met1-Xn-Met5-Xn-Met10 motif. The latter is also evident in the cyclic voltammetry studies of the Cu-BS complex. On the other hand, the Cu2+ binding features of the His site in both proteins, as modeled by AS(45-55) and BS(60-70), are identical, indicating that the shift in the His position does not affect its coordination features. Finally, replacement of Glu46 by Ala does not alter Cu2+ binding to the His site, suggesting that the familial PD E46K mutation would not impact copper-induced aggregation. While further studies of the redox activity of copper bound to His50 in AS are required to understand the role of this site in metal-mediated aggregation, our study contributes to a better understanding of the bioinorganic chemistry of PD.

The Stimulatory Effects of Intracellular α-Synuclein on Synaptic Transmission Are Attenuated by 2-Octahydroisoquinolin-2(1H)-ylethanamine.

α-Synuclein (αSyn) species can be detected in synaptic boutons, where they play a crucial role in the pathogenesis of Parkinson's Disease (PD). However, the effects of intracellular αSyn species on synaptic transmission have not been thoroughly studied. Here, using patch-clamp recordings in hippocampal neurons, we report that αSyn oligomers (αSynO), intracellularly delivered through the patch electrode, produced a fast and potent effect on synaptic transmission, causing a substantial increase in the frequency, amplitude and transferred charge of spontaneous synaptic currents. We also found an increase in the frequency of miniature synaptic currents, suggesting an effect located at the presynaptic site of the synapsis. Furthermore, our in silico approximation using docking analysis and molecular dynamics simulations showed an interaction between a previously described small anti-amyloid beta (Aß) molecule, termed M30 (2-octahydroisoquinolin-2(1H)-ylethanamine), with a central hydrophobic region of αSyn. In line with this finding, our empirical data aimed to obtain oligomerization states with thioflavin T (ThT) and Western blot (WB) indicated that M30 interfered with αSyn aggregation and decreased the formation of higher-molecular-weight species. Furthermore, the effect of αSynO on synaptic physiology was also antagonized by M30, resulting in a decrease in the frequency, amplitude, and charge transferred of synaptic currents. Overall, the present results show an excitatory effect of intracellular αSyn low molecular-weight species, not previously described, that are able to affect synaptic transmission, and the potential of a small neuroactive molecule to interfere with the aggregation process and the synaptic effect of αSyn, suggesting that M30 could be a potential therapeutic strategy for synucleinopathies.

Protein Misfolding and Aggregation: The Relatedness between Parkinson's Disease and Hepatic Endoplasmic Reticulum Storage Disorders.

Dysfunction of cellular homeostasis can lead to misfolding of proteins thus acquiring conformations prone to polymerization into pathological aggregates. This process is associated with several disorders, including neurodegenerative diseases, such as Parkinson's disease (PD), and endoplasmic reticulum storage disorders (ERSDs), like alpha-1-antitrypsin deficiency (AATD) and hereditary hypofibrinogenemia with hepatic storage (HHHS). Given the shared pathophysiological mechanisms involved in such conditions, it is necessary to deepen our understanding of the basic principles of misfolding and aggregation akin to these diseases which, although heterogeneous in symptomatology, present similarities that could lead to potential mutual treatments. Here, we review: (i) the pathological bases leading to misfolding and aggregation of proteins involved in PD, AATD, and HHHS: alpha-synuclein, alpha-1-antitrypsin, and fibrinogen, respectively, (ii) the evidence linking each protein aggregation to the stress mechanisms occurring in the endoplasmic reticulum (ER) of each pathology, (iii) a comparison of the mechanisms related to dysfunction of proteostasis and regulation of homeostasis between the diseases (such as the unfolded protein response and/or autophagy), (iv) and clinical perspectives regarding possible common treatments focused on improving the defensive responses to protein aggregation for diseases as different as PD, and ERSDs.

Green Tea Epigallocatechin-3-gallate (EGCG) Targeting Protein Misfolding in Drug Discovery for Neurodegenerative Diseases.

The potential to treat neurodegenerative diseases (NDs) of the major bioactive compound of green tea, epigallocatechin-3-gallate (EGCG), is well documented. Numerous findings now suggest that EGCG targets protein misfolding and aggregation, a common cause and pathological mechanism in many NDs. Several studies have shown that EGCG interacts with misfolded proteins such as amyloid beta-peptide (Aß), linked to Alzheimer's disease (AD), and α-synuclein, linked to Parkinson's disease (PD). To date, NDs constitute a serious public health problem, causing a financial burden for health care systems worldwide. Although current treatments provide symptomatic relief, they do not stop or even slow the progression of these devastating disorders. Therefore, there is an urgent need to develop effective drugs for these incurable ailments. It is expected that targeting protein misfolding can serve as a therapeutic strategy for many NDs since protein misfolding is a common cause of neurodegeneration. In this context, EGCG may offer great potential opportunities in drug discovery for NDs. Therefore, this review critically discusses the role of EGCG in NDs drug discovery and provides updated information on the scientific evidence that EGCG can potentially be used to treat many of these fatal brain disorders.

Brazilin Removes Toxic Alpha-Synuclein and Seeding Competent Assemblies from Parkinson Brain by Altering Conformational Equilibrium.

Alpha-synuclein (α-syn) fibrils, a major constituent of the neurotoxic Lewy Bodies in Parkinson's disease, form via nucleation dependent polymerization and can replicate by a seeding mechanism. Brazilin, a small molecule derived from red cedarwood trees in Brazil, has been shown to inhibit the fibrillogenesis of amyloid-beta (Aß) and α-syn as well as remodel mature fibrils and reduce cytotoxicity. Here we test the effects of Brazilin on both seeded and unseeded α-syn fibril formation and show that the natural polyphenol inhibits fibrillogenesis of α-syn by a unique mechanism that alters conformational equilibria in two separate points of the assembly mechanism: Brazilin preserves the natively unfolded state of α-syn by specifically binding to the compact conformation of the α-syn monomer. Brazilin also eliminates seeding competence of α-syn assemblies from Parkinson's disease patient brain tissue, and reduces toxicity of pre-formed assemblies in primary neurons by inducing the formation of large fibril clusters. Molecular docking of Brazilin shows the molecule to interact both with unfolded α-syn monomers and with the cross-ß sheet structure of α-syn fibrils. Our findings suggest that Brazilin has substantial potential as a neuroprotective and therapeutic agent for Parkinson's disease.

The Cytotoxic Effect of α-Synuclein Aggregates.

Parkinson's disease is a neurodegenerative disorder involving a functional protein, α-synuclein, whose primary function is related to vesicle trafficking. However, α-synuclein is prone to form aggregates, and these inclusions, known as Lewy bodies, are the hallmark of Parkinson's disease. α-synuclein can alter its conformation and acquire aggregating capacity, forming aggregates containing ß-sheets. This protein's pathogenic importance is based on its ability to form oligomers that impair synaptic transmission and neuronal function by increasing membrane permeability and altering homeostasis, generating a deleterious effect over cells. First, we establish that oligomers interfere with the mechanical properties of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) membrane, as demonstrated by nanoindentation curves. In contrast, nanoindentation revealed that the α-synuclein monomer's presence leads to a much more resistant lipid bilayer. Moreover, the oligomers' interaction with cell membranes can promote lactate dehydrogenase (LDH) release, suggesting the activation of cytotoxic events.

Prolyl Endopeptidase-Like Facilitates the α-Synuclein Aggregation Seeding, and This Effect Is Reverted by Serine Peptidase Inhibitor PMSF.

The aggregation of α-synuclein (α-Syn) is a characteristic of Parkinson's disease (PD). α-Syn oligomerization/aggregation is accelerated by the serine peptidase, prolyl oligopeptidase (POP). Factors that affect POP conformation, including most of its inhibitors and an impairing mutation in its active site, influence the acceleration of α-Syn aggregation resulting from the interaction of these proteins. It is noteworthy, however, that α-Syn is not cleaved by POP. Prolyl endopeptidase-like (PREPL) protein is structurally related to the serine peptidases belonging to the POP family. Based on the α-Syn-POP studies and knowing that PREPL may contribute to the regulation of synaptic vesicle exocytosis, when this protein can encounter α-Syn, we investigated the α-Syn-PREPL interaction. The binding of these two human proteins was observed with an apparent affinity constant of about 5.7 µM and, as in the α-Syn assays with POP, the presence of PREPL accelerated the oligomerization/aggregation events, with no α-Syn cleavage. Furthermore, despite this lack of hydrolytic cleavage, the serine peptidase active site inhibitor phenylmethylsulfonyl fluoride (PMSF) abolished the enhancement of the α-Syn aggregation by PREPL. Therefore, given the attention to POP inhibitors as potential drugs to treat synucleinopathies, the present data point to PREPL as another potential target to be explored for this purpose.

X1INH, an improved next-generation affinity-optimized hydrazonic ligand, attenuates abnormal copper(I)/copper(II)-α-Syn interactions and affects protein aggregation in a cellular model of synucleinopathy.

Although normal aging presents an accumulation of copper and iron in the brain, this becomes more relevant in neurodegeneration. α-Synuclein (α-Syn) misfolding has long been linked with the development of Parkinson's disease (PD). Copper binding promotes aggregation of α-Syn, as well as generalized oxidative stress. In this sense, the use of therapies that target metal dyshomeostasis has been in focus in the past years. Metal-Protein Attenuating Compounds (MPACs) are moderate chelators that aim at disrupting specific, abnormal metal-protein interactions. Our research group has now established that N-acylhydrazones compose a set of truly encouraging MPACs for the bioinorganic management of metal-enhanced aggregopathies. In the present work, a novel ligand, namely 1-methyl-1H-imidazole-2-carboxaldehyde isonicotinoyl hydrazone (X1INH), is reported. We describe solution studies on the interaction and affinity of this compound for copper(ii) ions showing that a fine tuning of metal-affinity was achieved. A series of in vitro biophysical NMR experiments were performed in order to assess the X1INH ability to compete with α-Syn monomers for the binding of both copper(i) and copper(ii) ions, which are central in PD pathology. A preference for copper(i) has been observed. X1INH is less toxic to human neuroglioma (H4) cells in comparison to structure-related compounds. Finally, we show that treatment with X1INH results in a higher number of smaller, less compact inclusions in a well-established model of α-Syn aggregation. Thus, X1INH constitutes a promising MPAC for the treatment of Parkinson's disease.

Order-disorder skewness in alpha-synuclein: a key mechanism to recognize membrane curvature.

Currently, membrane curvature is understood as an active mechanism to control cells spatial organization and activity. Protein processes involved in sensing and generating curvature are therefore of major interest. In this work, we have studied α-synuclein interactions with a model lipid bilayer, inducing curvature in a controlled manner and describing protein responses at molecular level. We show that the intrinsically disordered region of α-synuclein binds to the bilayer as an acknowledgment to the induced curvature, a mechanism used by the interacting protein-membrane assembly to relieve free energy. We have calculated free energies for bending the bilayer with α-synuclein adsorbed on the surface and we have established the crucial role of the intrinsically disordered region, suggesting that a dynamic order/disorder interplay takes place as the bilayer reorganizes to bend.

Brazilin Inhibits α-Synuclein Fibrillogenesis, Disrupts Mature Fibrils, and Protects against Amyloid-Induced Cytotoxicity.

The inhibitory effect of brazilin against α-synuclein (α-syn) fibrillogenesis, disruption effect against mature fibrils, and the following cytotoxicity were examined by systematical biochemical, biophysical, cellular biological, and molecular simulation experiments. It is found that brazilin inhibited α-syn fibrillogenesis and disrupted the performed fibrils with a concentration-dependent manner. Moreover, cellular experimental data showed that brazilin effectively reduced the cytotoxicity induced by α-syn aggregates. Finally, molecular dynamics simulations were performed to explore the interactions between brazilin and α-syn pentamer. It is found that brazilin directly interacts with α-syn pentamer, and the hydrophobic interactions are favorable for brazilin binding with the α-syn pentamer, while the electrostatic part provides adverse effects. Three binding regions were identified to inhibit α-syn fibrillogenesis or disrupt the preformed aggregates. Furthermore, six important residues (i.e., G51, V52, A53, E61, V66, and K80) of α-syn were also identified. We expected that brazilin is an effective agent against α-syn fibrillogenesis and associated cytotoxicity.

Metal coordination and peripheral substitution modulate the activity of cyclic tetrapyrroles on αS aggregation: a structural and cell-based study.

The discovery of aggregation inhibitors and the elucidation of their mechanism of action are key in the quest to mitigate the toxic consequences of amyloid formation. We have previously characterized the antiamyloidogenic mechanism of action of sodium phtalocyanine tetrasulfonate ([Na4(H2PcTS)]) on α-Synuclein (αS), demonstrating that specific aromatic interactions are fundamental for the inhibition of amyloid assembly. Here we studied the influence that metal preferential affinity and peripheral substituents may have on the activity of tetrapyrrolic compounds on αS aggregation. For the first time, our laboratory has extended the studies in the field of the bioinorganic chemistry and biophysics to cellular biology, using a well-established cell-based model to study αS aggregation. The interaction scenario described in our work revealed that both N- and C-terminal regions of αS represent binding interfaces for the studied compounds, a behavior that is mainly driven by the presence of negatively or positively charged substituents located at the periphery of the macrocycle. Binding modes of the tetrapyrrole ligands to αS are determined by the planarity and hydrophobicity of the aromatic ring system in the tetrapyrrolic molecule and/or the preferential affinity of the metal ion conjugated at the center of the macrocyclic ring. The different capability of phthalocyanines and meso-tetra (N-methyl-4-pyridyl) porphine tetrachloride ([H2PrTPCl4]) to modulate αS aggregation in vitro was reproduced in cell-based models of αS aggregation, demonstrating unequivocally that the modulation exerted by these compounds on amyloid assembly is a direct consequence of their interaction with the target protein.

Nitroalkylation of α-Synuclein by Nitro-Oleic Acid: Implications for Parkinson's Disease.

α-Synuclein (α-syn) represents the main component of the amyloid aggregates present in Parkinson's disease and other neurodegenerative disorders, collectively named synucleinopathies. Although α-syn is considered a natively unfolded protein, it shows great structural flexibility which allows the protein to adopt highly rich beta-sheet structures like protofibrils, oligomers and fibrils. In addition, this protein can adopt alpha-helix rich structures when interacts with fatty acids or acidic phospholipid vesicle membranes. When analyzing the toxicity of α-syn, protein oligomers are thought to be the main neurotoxic species by mechanisms that involve modification of intracellular calcium levels, mitochondrial and lysosomal function. Extracellular fibrillar α-syn promotes intracellular protein aggregation and shows many toxic effects as well. Nitro-fatty acids (nitroalkenes) represent novel pleiotropic anti-inflammatory signaling mediators that could interact with α-syn to exert unraveling actions. Herein, we demonstrated that nitro-oleic acid (NO2-OA) nitroalkylate α-syn, forming a covalent adduct at histidine-50. The nitroalkylated-α-syn exhibited strong affinity for phospholipid vesicles, moving the protein to the membrane compartment independent of composition of the membrane phospholipids. Moreover, NO2-OA-modified α-syn showed a reduced capacity to induce α-syn fibrillization compared to the non-nitrated oleic acid. From this data we hypothesize that nitroalkenes, in particular NO2-OA, may inhibit α-syn fibril formation exerting protective actions in Parkinson's disease.

Effects of alpha-synuclein post-translational modifications on metal binding.

Parkinson's disease is the second most common neurodegenerative disorder worldwide. Neurodegeneration in this pathology is characterized by the loss of dopaminergic neurons in the substantia nigra, coupled with cytoplasmic inclusions known as Lewy bodies containing α-synuclein. The brain is an organ that concentrates metal ions, and there is emerging evidence that a break-down in metal homeostasis may be a critical factor in a variety of neurodegenerative diseases. α-synuclein has emerged as an important metal-binding protein in the brain, whereas these interactions play an important role in its aggregation and might represent a link between protein aggregation, oxidative damage, and neuronal cell loss. Additionally, α-synuclein undergoes several post-translational modifications that regulate its structure and physiological function, and may be linked to the aggregation and/or oligomer formation. This review is focused on the interaction of this protein with physiologically relevant metal ions, highlighting the cases where metal-AS interactions profile as key modulators for its structural, aggregation, and membrane-binding properties. The impact of α-synuclein phosphorylation and N-terminal acetylation in the metal-binding properties of the protein are also discussed, underscoring a potential interplay between PTMs and metal ion binding in regulating α-synuclein physiological functions and its role in pathology. This article is part of the Special Issue "Synuclein".

Formation of large oligomers of DOPAL-modified α-synuclein is modulated by the oxidation of methionine residues located at C-terminal domain.

The formation of neurotoxic oligomers of the presynaptic protein α-Synuclein (aSyn) is suggested to be associated with Parkinson's disease neurodegeneration. In this respect, it was demonstrated that the aldehyde 3,4-dihydroxyphenylacetaldehyde (DOPAL), a product from the enzymatic oxidation of dopamine, is capable of stabilizing potentially toxic aSyn oligomers via formation of covalent adducts with Lys residues of the protein. In addition, DOPAL-induced production of reactive oxygen species (ROS) leads to the oxidation of aSyn's Met residues to Met-sulfoxide. Recently, our group pointed out that the pre-oxidation of all-four Met residues of aSyn, upon treatment with H2O2, decreases the formation of large aSyn-DOPAL oligomers, which are suggested to be more toxic to neurons than the corresponding small oligomers (Carmo-Gonçalves et al., Biochem. Biophys. Res. Comm. 505, 295-301. 2018). By using a series of Met to Val mutants of aSyn, we demonstrated that the ability of aSyn to scavenge ROS/H2O2 generated from DOPAL oxidation is primarily dependent on Met residues located at the C-terminal domain of the protein, which contrasts with the reactivity of aSyn against H2O2 itself in which N-terminal Met residues (notably Met5) were more readily oxidized. Interestingly, the substitution of C-terminal Met residues (particularly Met127) by Val increased the formation of DOPAL-induced large oligomers in comparison with the wild-type protein. In this context, we demonstrated that the hydrophobicity of aSyn monomer, which is affected distinctively by the oxidation of N- versus C-terminal methionines, is correlated with the formation of large (but not small) oligomers of aSyn mediated by DOPAL.

Exploring the role of methionine residues on the oligomerization and neurotoxic properties of DOPAL-modified α-synuclein.

The dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) is believed to play a central role in Parkinson's disease neurodegeneration by stabilizing potentially toxic oligomers of the presynaptic protein α-Synuclein (aSyn). Besides the formation of covalent DOPAL-Lys adducts, DOPAL promotes the oxidation of Met residues of aSyn, which is also a common oxidative post-translational modification found in the protein in vivo. Herein we set out to address the role of Met residues on the oligomerization and neurotoxic properties of DOPAL-modified aSyn. Our data indicate that DOPAL promotes the formation of two distinct types of aSyn oligomers: large and small (dimer and trimers) oligomers, which seem to be generated by independent mechanisms and cannot be interconverted by using denaturing agents. Interestingly, H2O2-treated aSyn monomer, which exhibits all-four Met residues oxidized to Met-sulfoxide, exhibited a reduced ability to form large oligomers upon treatment with DOPAL, with no effect on the population of small oligomers. In this context, triple Met-Val mutant M5V/M116V/M127V exhibited an increased population of large aSyn-DOPAL oligomers in comparison with the wild-type protein. Interestingly, the stabilization of large rather than small oligomers seems to be associated with an enhanced toxicity of DOPAL-aSyn adducts. Collectively, these findings indicate that Met residues may play an important role in modulating both the oligomerization and the neurotoxic properties of DOPAL-derived aSyn species.