Protein interactions and misfolding analyzed by AFM force spectroscopy

Protein misfolding is conformational transition dramatically facilitating the assembly of protein molecules into aggregates of various morphologies. Spontaneous formation of specific aggregates, mostly amyloid fibrils, was initially believed to be limited to proteins involved in the development of amyloidoses. However, recent studies show that, depending on conditions, the majority of proteins undergo structural transitions leading to the appearance of amyloidogenic intermediates followed by aggregate formation. Various techniques have been used to characterize the protein misfolding facilitating the aggregation process, but no direct evidence as to how such a conformational transition increases the intermolecular interactions has been obtained as of yet. We have applied atomic force microscopy (AFM) to follow the interaction between protein molecules as a function of pH. These studies were performed for three unrelated and structurally distinctive proteins, alpha-synuclein, amyloid beta-peptide (Abeta) and lysozyme. It was shown that the attractive force between homologous protein molecules is minimal at physiological pH and increases dramatically at acidic pH. Moreover, the dependence of the pulling forces is sharp, suggesting a pH-dependent conformational transition within the protein. Parallel circular dichroism (CD) measurements performed for alpha-synuclein and Abeta revealed that the decrease in pH is accompanied by a sharp conformational transition from a random coil at neutral pH to the more ordered, predominantly beta-sheet, structure at low pH. Importantly, the pH ranges for these conformational transitions coincide with those of pulling forces changes detected by AFM. In addition, protein self-assembly into filamentous aggregates studied by AFM imaging was shown to be facilitated at pH values corresponding to the maximum of pulling forces. Overall, these results indicate that proteins at acidic pH undergo structural transition into conformations responsible for the dramatic increase in interprotein interaction and promoting the formation of protein aggregates.     

The Hidden Cell-to-Cell Trail of α-Synuclein Aggregates

The progressive accumulation of insoluble aggregates of the presynaptic protein alpha-synuclein (α-Syn) is a hallmark of neurodegenerative disorders including Parkinson's disease (PD), Multiple System Atrophy, and Dementia with Lewy Bodies, commonly referred to as synucleinopathies. Despite considerable progress on the structural biology of these aggregates, the molecular mechanisms mediating their cell-to-cell transmission, propagation, and neurotoxicity remain only partially understood. Numerous studies have highlighted the stereotypical spatiotemporal spreading of pathological α-Syn aggregates across different tissues and anatomically connected brain regions over time. Experimental evidence from various cellular and animal models indicate that α-Syn transfer occurs in two defined steps: the release of pathogenic α-Syn species from infected cells, and their uptake via passive or active endocytic pathways. Once α-Syn aggregates have been internalized, little is known about what drives their toxicity or how they interact with the endogenous protein to promote its misfolding and subsequent aggregation. Similarly, unknown genetic factors modulate different cellular responses to the aggregation and accumulation of pathogenic α-Syn species. Here we discuss the current understanding of the molecular phenomena associated with the intercellular spreading of pathogenic α-Syn seeds and summarize the evidence supporting the transmission hypothesis. Understanding the molecular mechanisms involved in α-Syn aggregates transmission is essential to develop novel targeted therapeutics against PD and related synucleinopathies.     

Assays for α-synuclein aggregation

This review describes different ways to achieve and monitor reproducible aggregation of α-synuclein, a key protein in the development of Parkinson's disease. For most globular proteins, aggregation is promoted by partially denaturing conditions which compromise the native state without destabilizing the intermolecular contacts required for accumulation of regular amyloid structure. As a natively disordered protein, α-synuclein can fibrillate under physiological conditions and this process is actually stimulated by conditions that promote structure formation, such as low pH, ions, polyamines, anionic surfactants, fluorinated alcohols and agitation. Reproducibility is a critical issue since α-synuclein shows erratic fibrillation behavior on its own. Agitation in combination with glass beads significantly reduces the variability of aggregation time curves, but the most reproducible aggregation is achieved by sub-micellar concentrations of SDS, which promote the rapid formation of small clusters of α-synuclein around shared micelles. Although the fibrils produced this way have a different appearance and secondary structure, they are rich in cross-β structure and are amenable to high-throughput screening assays. Although such assays at best provide a very simplistic recapitulation of physiological conditions, they allow the investigator to focus on well-defined molecular events and may provide the opportunity to identify, e.g. small molecule inhibitors of aggregation that affect these steps. Subsequent experiments in more complex cellular and whole-organism environments can then validate whether there is any relation between these molecular interactions and the broader biological context.     

Multifaceted interactions mediated by intrinsically disordered regions play key roles in alpha synuclein aggregation

The aggregation of Alpha Synuclein (α-Syn) into fibrils is associated with the pathology of several neurodegenerative diseases. Pathologic aggregates of α-Syn adopt multiple fibril topologies and are known to be transferred between cells via templated seeding. Monomeric α-Syn is an intrinsically disordered protein (IDP) with amphiphilic N-terminal, hydrophobic-central, and negatively charged C-terminal domains. Here, we review recent work elucidating the mechanism of α-Syn aggregation and identify the key and multifaceted roles played by the N- and C-terminal domains in the initiation and growth of aggregates as well as in the templated seeding involved in cell-to-cell propagation. The charge content of the C-terminal domain, which is sensitive to environmental conditions like organelle pH, is a key regulator of intermolecular interactions involved in fibril growth and templated propagation. An appreciation of the complex and multifaceted roles played by the intrinsically disordered terminal domains suggests novel opportunities for the development of potent inhibitors against synucleinopathies.     

α-Synuclein misfolding and aggregation: Implications in Parkinson’s disease pathogenesis

α-Synuclein (α-Syn) has been extensively studied for its structural and biophysical properties owing to its pathophysiological role in Parkinson’s disease (PD). Lewy bodies and Lewy neurites are the pathological hallmarks of PD and contain α-Syn aggregates as their major component. It was therefore hypothesized that α-Syn aggregation is actively associated with PD pathogenesis. The central role of α-Syn aggregation in PD is further supported by the identification of point mutations in α-Syn protein associated with rare familial forms of PD. However, the correlation between aggregation propensities of α-Syn mutants and their association with PD phenotype is not straightforward. Recent evidence suggested that oligomers, formed during the initial stages of aggregation, are the potent neurotoxic species causing cell death in PD. However, the heterogeneous and unstable nature of these oligomers limit their detailed characterization. α-Syn fibrils, on the contrary, are shown to be the infectious agents and propagate in a prion-like manner. Although α-Syn is an intrinsically disordered protein, it exhibits remarkable conformational plasticity by adopting a range of structural conformations under different environmental conditions. In this review, we focus on the structural and functional aspects of α-Syn and role of potential factors that may contribute to the underlying mechanism of synucleinopathies. This information will help to identify novel targets and develop specific therapeutic strategies to combat Parkinson’s and other protein aggregation related neurodegenerative diseases.     

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.     

Deciphering the robustness of pyrazolo-pyridine carboxylate core structure-based compounds for inhibiting α-synuclein in transgenic C. elegans model of Synucleinopathy

Parkinson’s disease (PD), a calamitous neurodegenerative disorder with no cure till date, is closely allied with the misfolding and aggregation of α-Synuclein (α -Syn). Inhibition of α-Syn aggregation is one of the optimistic approaches for the treatment for PD. Here, we carried out hypothesis-driven studies towards synthesising a series of pyrazolo-pyridine carboxylate containing compounds (7a–7m) targeted at reducing deleterious α-Syn aggregation. The target compounds were synthesized through multi-step organic synthesis reactions. From docking studies, compounds 7b, 7g and 7i displayed better interaction with the key residues of α-Syn with values: −6.8, −8.9 and −7.2 Kcal/mol, respectively. In vivo transgenic C. elegans model of Synucleinopathy was used to evaluate the ability of the designed and synthesized compounds to inhibit α-Syn aggregation. These lead compounds 7b, 7g and 7i displayed 1.7, 2.4 and 1.5-fold inhibition of α-Syn with respect to the control. Further, the strategy of employing pyrazolo-pyridine-based compounds worked with success and these scaffolds could be further modified and validated for betterment of endpoints associated with PD.     

Methionine oxidation stabilizes non-toxic oligomers of α-synuclein through strengthening the auto-inhibitory intra-molecular long-range interactions

Oxidative stress and aggregation of the presynaptic protein α-synuclein (α-Syn) are implied in the pathogenesis of Parkinson's disease and several other neurodegenerative diseases. Various posttranslational modifications, such as oxidation, nitration and truncation, have significant effects on the kinetics of α-Syn fibrillation in vitro. α-Syn is a typical natively unfolded protein, which possesses some residual structure. The existence of long-range intra-molecular interactions between the C-terminal tail (residues 120–140) and the central part of α-Syn (residues 30–100) was recently established (Bertoncini et al. (2005) Proc Natl Acad Sci U S A 102, 1430–1435). Since α-Syn has four methionines, two of which (Met 1 and 5) are at the N-terminus and the other two (Met 116 and 127) are in the hydrophobic cluster at the C-terminus of protein, the perturbation of these residues via their oxidation represents a good model for studying the effect of long-range interaction on α-Syn fibril formation. In this paper we show that Met 1, 116, and 127 are more protected from the oxidation than Met 5 likely due to the residual structure in the natively unfolded α-Syn. In addition to the hydrophobic interactions between the C-terminal hydrophobic cluster and hydrophobic central region of α-Syn, there are some long-range electrostatic interactions in this protein. Both of these interactions likely serve as auto-inhibitors of α-Syn fibrillation. Methionine oxidation affects both electrostatic and hydrophobic long-range interactions in α-Syn. Finally, oxidation of methionines by H₂O₂ greatly inhibited α-Syn fibrillation in vitro, leading to the formation of relatively stable oligomers, which are not toxic to dopaminergic and GABAergic neurons.     

Hsc70 protein interaction with soluble and fibrillar alpha-synuclein

The aggregation of α-synuclein (α-Syn), the primary component of Lewy bodies, into high molecular weight assemblies is strongly associated with Parkinson disease. This event is believed to result from a conformational change within native α-Syn. Molecular chaperones exert critical housekeeping functions in vivo including refolding, maintaining in a soluble state, and/or pacifying protein aggregates. The influence of the stress-induced heat shock protein 70 (Hsp70) on α-Syn aggregation has been notably investigated. The constitutively expressed chaperone Hsc70 acts as an antiaggregation barrier before cells are overwhelmed with α-Syn aggregates and Hsp70 expression induced. Here, we investigate the interaction between Hsc70 and α-Syn, the consequences of this interaction, and the role of nucleotides and co-chaperones Hdj1 and Hdj2 as modulators. We show that Hsc70 sequesters soluble α-Syn in an assembly incompetent complex in the absence of ATP. The affinity of Hsc70 for soluble α-Syn diminishes upon addition of ATP alone or together with its co-chaperones Hdj1 or Hdj2 allowing faster binding and release of client proteins thus abolishing α-Syn assembly inhibition by Hsc70. We show that Hsc70 binds α-Syn fibrils with a 5-fold tighter affinity compared with soluble α-Syn. This suggests that Hsc70 preferentially interacts with high molecular weight α-Syn assemblies in vivo. Hsc70 binding certainly has an impact on the physicochemical properties of α-Syn assemblies. We show a reduced cellular toxicity of α-Syn fibrils coated with Hsc70 compared with "naked" fibrils. Hsc70 may therefore significantly affect the cellular propagation of α-Syn aggregates and their spread throughout the central nervous system in Parkinson disease.     

Differential copper binding to alpha-synuclein and its disease-associated mutants affect the aggregation and amyloid formation

BackgroundCopper is an essential trace element required for the proper functioning of various enzymes present in the central nervous system. An imbalance in the copper homeostasis results in the pathology of various neurodegenerative disorders including Parkinson’s Disease. Hence, residue specific interaction of Cu²⁺ to α-Syn along with the familial mutants H50Q and G51D needs to be studied in detail.MethodsWe investigated the residue specific mapping of Cu²⁺ binding sites and binding strength using solution-state NMR and ITC respectively. The aggregation kinetics, secondary structural changes, and morphology of the formed fibrils in the presence and absence of Cu²⁺ were studied using fluorescence, CD, and AFM respectively.ResultsCopper binding to α-Syn takes place at three different sites with a higher affinity for the region 48-53. While one of the sites got abolished in the case of H50Q, the mutant G51D showed a binding pattern similar to WT. The aggregation kinetics of these proteins in the presence of Cu²⁺ showed an enhanced rate of fibril formation with a pronounced effect for G51D.ConclusionCu²⁺ binding results in the destabilization of long-range tertiary interactions in α-Syn leading to the exposure of highly amyloidogenic NAC region which results in the increased rate of fibril formation. Although the residues 48-53 have a stronger affinity for Cu²⁺ in case of WT and G51D, the binding is not responsible for enhancing the rate of fibril formation in case of H50Q.General SignificanceThese findings will help in the better understanding of Cu²⁺ catalyzed aggregation of synucleins.     

Pathological implications of nucleic acid interactions with proteins associated with neurodegenerative diseases

Protein misfolding disorders (PMDs) refer to a group of diseases related to the misfolding of particular proteins that aggregate and deposit in the cells and tissues of humans and other mammals. The mechanisms that trigger protein misfolding and aggregation are still not fully understood. Increasing experimental evidence indicates that abnormal interactions between PMD-related proteins and nucleic acids (NAs) can induce conformational changes. Here, we discuss these protein–NA interactions and address the role of deoxyribonucleic (DNA) and ribonucleic (RNA) acid molecules in the conformational conversion of different proteins that aggregate in PMDs, such as Alzheimer’s, Parkinson’s, and prion diseases. Studies on the affinity, stability, and specificity of proteins involved in neurodegenerative diseases and NAs are specifically addressed. A landscape of reciprocal effects resulting from the binding of prion proteins, amyloid-β peptides, tau proteins, huntingtin, and α-synuclein are presented here to clarify the possible role of NAs, not only as encoders of genetic information but also in triggering PMDs.     

alpha-Synuclein misfolding: single molecule AFM force spectroscopy study

Protein misfolding and aggregation are the very first and critical steps in development of various neurodegenerative disorders, including Parkinson’s disease, induced by misfolding of α-synuclein. Thus, elucidating properties of proteins in misfolded states and understanding the mechanisms of their assembly into the disease prone aggregates are critical for the development of rational approaches to prevent protein misfolding-mediated pathologies. To accomplish this goal and as a first step to elucidate the mechanism of α-synuclein misfolding, we applied single-molecule force spectroscopy capable of detecting protein misfolding. We immobilized α-synuclein molecules at their C-termini at the atomic force microscope tips and substrate surfaces, and measured the interaction between the proteins by probing the microscope tip at various locations on the surface. Using this approach, we detected α-synuclein misfolded states by enhanced interprotein interaction. We used a dynamics force spectroscopy approach to measure such an important characteristic of dimers of misfolded α-synuclein as their lifetimes. We found that the dimer lifetimes are in the range of seconds and these values are much higher than the characteristics for the dynamics of the protein in monomeric state. These data show that compared to highly dynamic monomeric forms, α-synuclein dimers are much more stable and thus can serve as stable nuclei for the formation of multimeric and aggregated forms of α-synuclein. Importantly, two different lifetimes were observed for the dimers, suggesting that aggregation can follow different pathways that may lead to different aggregated morphologies of α-synuclein.     

At Low Concentrations, 3,4-Dihydroxyphenylacetic Acid (DOPAC) Binds Non-Covalently to α-Synuclein and Prevents Its Fibrillation

Several studies have shown that catecholamines can inhibit the fibrillation of α-synuclein (α-Syn), a small presynaptic protein whose aggregation is believed to be a critical step in the etiology of Parkinson's disease and several other neurodegenerative disorders. However, the mechanism of this inhibition is uncertain. We show here that substoichiometric concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC), a normal product of the metabolism of dopamine, can inhibit the fibrillation of α-Syn, due to non-covalent binding of DOPAC to α-Syn monomer. Intriguingly, the presence of α-Syn accelerates the spontaneous oxidation of DOPAC, and the oxidized form of DOPAC (the quinone) is responsible for the fibrillation inhibition. In addition, the presence of DOPAC leads to the oxidation of the methionine residues of α-Syn, probably due to the H₂O₂ production as a by-product of DOPAC oxidation. The lack of fibrillation results from the formation of stable oligomers, which are very similar to those observed transiently at early stages of the α-Syn fibrillation. A possible explanation for this phenomenon is that DOPAC stabilizes the normally transient oligomers and prevents them from subsequent fibril formation. The analysis of the α-Syn Y39W variant suggests that DOPAC binds non-covalently to the same N-terminal region of α-Syn as lipid vesicles, probably in the vicinity of residue 39. In contrast to the compounds with 1,2-dihydroxyphenyl groups (DOPAC and catechol), their 1,4-dihydroxyphenyl isomers (hydroquinone and homogentisic acid) are able to modify α-Syn covalently, probably due to the less steric hindrance in the Michael addition.     

Hsp31, a member of the DJ-1 superfamily,is a multitasking stress responder with chaperone activity

Among different types of protein aggregation, amyloids are a biochemically well characterized state of protein aggregation that are associated with a large number of neurodegenerative diseases including Parkinson's disease, Alzheimer and Creutzfeldt-Jakob disease. Yeast, Saccharomyces cerevisiae is an insightful model to understand the underlying mechanism of protein aggregation. Many yeast molecular chaperones can modulate aggregation and misfolding of proteins including α-Syn and the Sup35 prion. Hsp31 is a homodimeric protein structurally similar to human DJ-1, a Parkinson's disease-linked protein, and both are members of the DJ-1/ThiJ/PfpI superfamily. An emerging view is that Hsp31 and its associated superfamily members each have divergent multitasking functions that have the common theme of responding and managing various types of cellular stress. Hsp31 has several biochemical activities including chaperone and detoxifying enzyme activities that modulate at various points of a stress pathway such as toxicity associated with protein misfolding. However, we have shown the protective role of Hsp31's chaperone activity can operate independent of detoxifying enzyme activities in preventing the early stages of protein aggregate formation and associated cellular toxicities. We provide additional data that collectively supports the multiple functional roles that can be accomplished independent of each other. We present data indicating Hsp31 purified from yeast is more active compared to expression and purification from E. coli suggesting that posttranslational modifications could be important for Hsp31 to be fully active. We also compare the similarities and differences in activities among paralogs of Hsp31 supporting a model in which this protein family has overlapping but diverging roles in responding to various sources of cellular stresses.     

Effect of polyols on the structure and aggregation of recombinant human γ-Synuclein,an intrinsically disordered protein

Polyol osmolytes accumulated in cells under stress are known to promote stability in globular proteins with respect to their increasing hydroxyl groups but their effect on the structure, stability and aggregation of intrinsically disordered proteins (IDPs) is still elusive. The lack of a natively folded structure in intrinsically disordered proteins under physiological conditions results in their aggregation and fibrillation that gives rise to a number of diseases. We have investigated the effect of a series of polyols, ethylene glycol (EG), glycerol, erythritol, xylitol and sorbitol on the fibrillation pathway of recombinant human γ-Synuclein, used as a model, for an IDP known to form fibrils that play a role in neurodegeneration and cancer. With an increase in the number of –OH groups in polyols except EG, we observe a decrease in lag time for fibrillation at equimolar concentrations, suggesting stronger preferential exclusion of polyols that promotes γ-Syn self-association and oligomerization. The polyols act early during nucleation and their diverse effect on the rate of fibrillation suggests the role of favourable solvent-side chain interactions. With increasing –OH group, polyols stabilize the natively unfolded conformation of γ-Syn under non-fibrillating conditions and delay the structural transition to characteristic β-sheet structure by forming an α-helical intermediate during fibrillation. The results, overall suggest that the effect of osmolytes on IDPs is much more complex than their effect on globular protein stability and aggregation and a fine balance between the dominant unfavourable osmolyte-peptide backbone and favourable osmolyte-charged side chain interactions would govern their stability and aggregation properties.     

Single-molecule atomic force microscopy force spectroscopy study of Aβ-40 interactions

Misfolding and aggregation of amyloid β-40 (Aβ-40) peptide play key roles in the development of Alzheimer's disease (AD). However, very little is known about the molecular mechanisms underlying these molecular processes. We developed a novel experimental approach that can directly probe aggregation-prone states of proteins and their interactions. In this approach, the proteins are anchored to the surface of the atomic force microscopy substrate (mica) and the probe, and the interaction between anchored molecules is measured in the approach-retraction cycles. We used dynamic force spectroscopy (DFS) to measure the stability of transiently formed dimers. One of the major findings from DFS analysis of α-synuclein (α-Syn) is that dimeric complexes formed by misfolded α-Syn protein are very stable and dissociate over a range of seconds. This differs markedly from the dynamics of monomers, which occurs on a microsecond to nanosecond time scale. Here we applied the same approach to quantitatively characterize interactions of Aβ-40 peptides over a broad range of pH values. These studies showed that misfolded dimers are characterized by lifetimes in the range of seconds. This value depends on pH and varies between 2.7 s for pH 2.7 and 0.1 s for pH 7, indicating that the aggregation properties of Aβ-40 are modulated by the environmental conditions. The analysis of the contour lengths revealed the existence of various pathways for dimer dissociation, suggesting that dimers with different conformations are formed. These structural variations result in different aggregation pathways, leading to different types of oligomers and higher-order aggregates, including fibrils.     

Effect of Trehalose on PC12 Cells Overexpressing Wild-Type or A53T Mutant α-synuclein

Accumulation of α-synuclein (α-Syn) is a common pathology for both familiar and sporadic Parkinson's disease (PD), enhancing its clearance might be a promising strategy for treating PD. To assess the potential of trehalose in this regard, we investigated its effect on the PC12 cells overexpressing wild type (WT) or A53T mutant α-Syn and the implicated pathway it might mediated. We observed that trehalose promoted the clearance of A53T α-Syn but not WT α-Syn in PC12 cells, and confirmed the increased LC3 and Lysotracker RED positive autolysosomes by using lysotracker and LC3 staining, the enhanced expression of LC3-II in Western blot, and more autophagosomes under Transmission Electron Microscope in a dose dependent manner after the trehalose treatment. The activation of autophagy can be alleviated by applying macroautophagy inhibitor 3-methyladenine (3-MA). In addition, degradation of A53T and WT α-Syn was blocked after Ubiquitin Proteasome System (UPS) inhibitor (MG132) was applied in those PC12 cells overexpressing A53T or WT α-Syn, suggesting that A53T α-Syn could be degraded by both UPS and macroautophagy. But the effect of trehalose on A53T α-Syn is mainly mediated through the macroautophagy pathway, which is not a dominant way for WT α-Syn clearance. Further in vivo research will be needed to verify the effectiveness of trehalose in treating PD.     

Study of Cosolvent-Induced α-Chymotrypsin Fibrillogenesis: Does Protein Surface Hydrophobicity Trigger Early Stages of Aggregation Reaction?

The misfolding of specific proteins is often associated with their assembly into fibrillar aggregates, commonly termed amyloid fibrils. Despite the many efforts expended to characterize amyloid formation in vitro, there is no deep knowledge about the environment (in which aggregation occurs) as well as mechanism of this type of protein aggregation. Alpha-chymotrypsin was recently driven toward amyloid aggregation by the addition of intermediate concentrations of trifluoroethanol. In the present study, approaches such as turbidimetric, thermodynamic, intrinsic fluorescence and quenching studies as well as chemical modification have been successfully used to elucidate the underlying role of hydrophobic interactions (involved in early stages of amyloid formation) in α-chymotrypsin-based experimental system.     

Inhibition of formation of α-synuclein inclusions by mannosylglycerate in a yeast model of Parkinson's disease

Background

Protein aggregation in the brain is a central hallmark in many neurodegenerative diseases. In Parkinson's disease, α-synuclein (α-Syn) is the major component of the intraneuronal inclusions found in the brains of patients. Current therapeutics is merely symptomatic, and there is a pressing need for developing novel therapies. Previously we showed that mannosylglycerate (MG), a compatible solute typical of marine microorganisms thriving in hot environments, is highly effective in protecting a variety of model proteins against thermal denaturation and aggregation in vitro.

Methods

Saccharomyces cerevisiae cells expressing eGFP-tagged α-Syn, were further engineered to synthesize MG. The number of cells with fluorescent foci was assessed by fluorescence microscopy. Fluorescence spectroscopy and transmission electron microscopy were used to monitor fibril formation in vitro.

Results

We observed a 3.3-fold reduction in the number of cells with α-Syn foci and mild attenuation of α-Syn-induced toxicity. Accordingly, sucrose gradient analysis confirmed a clear reduction in the size-range of α-Syn species in the cells. MG did not affect the expression levels of α-Syn or its degradation rate. Moreover, MG did not induce molecular chaperones (Hsp104, Hsp70 and Hsp40), suggesting the implication of other mechanisms for α-Syn stabilization. MG also inhibited α-Syn fibrillation in vitro.

Conclusions

MG acts as a chemical chaperone and the stabilization mechanism involves direct solute/protein interactions.

General significance

This is the first demonstration of the anti-aggregating ability of MG in the intracellular milieu. The work shows that MG is a good candidate to inspire the development of new drugs for protein-misfolding diseases.     

Structural and mechanistic insights into modulation of α-Synuclein fibril formation by aloin and emodin

α-Synuclein (α-Syn) aggregation/fibrillation is a leading cause of neuronal death and is one of the major pathogenic factors involved in the progression of Parkinson's' disease (PD). Against this backdrop, discovering new molecules as inhibitors or modulators of α-Syn aggregation/fibrillation is a subject of enormous research. In this study, we have shown modulation, disaggregation, and neuroprotective potential of aloin and emodin against α-Syn aggregation/fibrillation. Thioflavin T (ThT) fluorescence assay showed an increase in lag phase from (51.14 ± 2) h to (68.58 ± 2) h and (74.14 ± 3) h in the presence of aloin and emodin respectively. ANS binding assay represents a modulatory effect of these molecules on hydrophobicity which is crucial for aggregates/fibril formation. NMR spectroscopy and tyrosine quenching studies reveal the binding of aloin/emodin with monomeric α-Syn. TEM and DLS micrographs illustrate the attenuating effect of aloin/emodin against the development of large aggregates/fibrils. Our seeding experiments suggest aloin/emodin generate seeding incompetent oligomers that direct the off-pathway aggregation/fibrillation. Also, aloin/emodin capably reduces the fibrils-induced cytotoxicity and disassembles the preexisting amyloid fibrils. These findings provide deep insight into the modulatory mechanism of α-Syn aggregation/fibrillation in the presence of aloin and emodin, thereby suggesting their potential roles as promising therapeutic molecules against aggregation/fibrillation related disorders.