Acceleration of alpha-synuclein aggregation by homologous peptides

alpha-Synuclein (alpha-Syn), amyloid beta-protein and prion protein are among the amyloidogenic proteins that are associated with the neurodegenerative diseases. These three proteins share a homologous region with a consensus sequence mainly consisting of glycine, alanine and valine residues (accordingly named as the GAV motif), which was proposed to be the critical core for the fibrillization and cytotoxicity. To understand the role of the GAV motif in protein amyloidogenesis, we studied the effects of the homologous peptides corresponding to the sequence of GAV motif region (residues 66-74) on alpha-Syn aggregation. The result shows that these peptides can promote fibrillization of wild-type alpha-Syn and induce that of the charge-incorporated mutants but not the GAV-deficient alpha-Syn mutant. The acceleration of alpha-Syn aggregation by the homologous peptides is under a sequence-specific manner. The interplay between the GAV peptide and the core regions in alpha-Syn may accelerate the aggregation process and stabilize the fibrils. This finding provides clues for developing peptide mimics that could promote transforming the toxic oligomers or protofibrils into the inert mature fibrils.     

Sensitive fluorescence polarization technique for rapid screening of alpha-synuclein oligomerization/fibrillization inhibitors

Parkinson's disease (PD) is characterized by the accumulation of fibrillar alpha-synuclein (alpha-Syn) inclusions known as Lewy bodies (LBs) and Lewy neurites. Mutations in the alpha-Syn gene or extra copies thereof cause familial PD or dementia with LBs (DLB) in rare kindreds, but abnormal accumulations of wildtype alpha-Syn also are implicated in the pathogenesis of sporadic PD, the most common movement disorder. Insights into mechanisms underlying alpha-Syn mediated neurodegeneration link alpha-Syn oligomerization and fibrillization to the onset and progression of PD. Thus, inhibiting alpha-Syn oligomer or fibril formation is a compelling target for discovering disease modifying therapies for PD, DLB, and related synucleinopathies. Although amyloid dyes recognize alpha-Syn fibrils, efficient detection of soluble oligomers remains a challenge. Here, we report a novel fluorescence polarization (FP) technique for examining alpha-Syn assembly by monitoring changes in its relative molecular mass during progression of normal alpha-Syn from highly soluble monomers to higher order multimers and thence insoluble amyloid fibrils. We report that FP is more sensitive than conventional amyloid dye methods for the quantification of mature fibrils, and that FP is capable of detecting oligomeric alpha-Syn, allowing for rapid automated screening of potential inhibitors of alpha-Syn oligomerization and fibrillization. Furthermore, FP can be combined with an amyloid dye in a single assay that simultaneously provides two independent biophysical readouts for monitoring alpha-Syn fibrillization. Thus, this FP method holds potential to accelerate discovery of disease modifying therapies for LB PD, DLB, and related neurodegenerative synucleinopathies.     

Inhibition of alpha-synuclein fibrillization by dopamine analogs via reaction with the amino groups of alpha-synuclein. Implication for dopaminergic neurodegeneration

Fibrillization of alpha-synuclein (alpha-Syn) is closely associated with the formation of Lewy bodies in neurons and dopamine (DA) is a potent inhibitor for the process, which is implicated in the causative pathogenesis of Parkinson's disease (PD). To elucidate any molecular mechanism that may have biological relevance, we tested the inhibitory abilities of DA and several analogs including chemically synthetic and natural polyphenols in vitro. The MS and NMR characterizations strongly demonstrate that DA and its analogs inhibit alpha-Syn fibrillization by a mechanism where the oxidation products (quinones) of DA analogs react with the amino groups of alpha-Syn chain, generating alpha-Syn-quinone adducts. It is likely that the amino groups of alpha-Syn undergo nucleophilic attack on the quinone moiety of DA analogs to form imino bonds. The covalently cross-linked alpha-Syn adducts by DA are primarily large molecular mass oligomers, while those by catechol and p-benzoquinone (or hydroquinone) are largely monomers or dimers. The DA quinoprotein retains the same cytotoxicity as the intact alpha-Syn, suggesting that the oligomeric intermediates are the major elements that are toxic to the neuronal cells. This finding implies that the reaction of alpha-Syn with DA is relevant to the selective dopaminergic loss in PD.     

Characterization of two VQIXXK motifs for tau fibrillization in vitro

Tau proteins are building blocks of the filaments that form neurofibrillary tangles of Alzheimer's disease (AD) and related neurodegenerative tauopathies. It was recently reported that two VQIXXK motifs in the microtubule (MT) binding region, named PHF6 and PHF6*, are responsible for tau fibrillization. However, the exact role each of these motifs plays in this process has not been analyzed in detail. Using a recombinant human tau fragment containing only the four MT-binding repeats (K18), we show that deletion of either PHF6 or PHF6* affected tau assembly but only PHF6 is essential for filament formation, suggesting a critical role of this motif. To determine the amino acid residues within PHF6 that are required for tau fibrillization, a series of deletion and mutation constructs targeting this motif were generated. Deletion of VQI in either PHF6 or PHF6* lessened but did not eliminate K18 fibrillization. However, removal of the single K311 residue from PHF6 completely abrogated the fibril formation of K18. K311D mutation of K18 inhibited tau filament formation, while K311A and K311R mutations had no effect. These data imply that charge change at position 311 is important in tau fibril formation. A similar requirement of nonnegative charge at this position for fibrillization was observed with the full-length human tau isoform (T40), and data from these studies indicate that the formation of fibrils by T40K311D and T40K311P mutants is repressed at the nucleation phase. These findings provide important insights into the mechanisms of tau fibrillization and suggest targets for AD drug discovery to ameliorate neurodegeneration mediated by filamentous tau pathologies.     

Hsp104 targets multiple intermediates on the amyloid pathway and suppresses the seeding capacity of Abeta fibrils and protofibrils

The heat shock protein Hsp104 has been reported to possess the ability to modulate protein aggregation and toxicity and to “catalyze” the disaggregation and recovery of protein aggregates, including amyloid fibrils, in yeast, Escherichia coli, mammalian cell cultures, and animal models of Huntington's disease and Parkinson's disease. To provide mechanistic insight into the molecular mechanisms by which Hsp104 modulates aggregation and fibrillogenesis, the effect of Hsp104 on the fibrillogenesis of amyloid beta (Aβ) was investigated by characterizing its ability to interfere with oligomerization and fibrillogenesis of different species along the amyloid-formation pathway of Aβ. To probe the disaggregation activity of Hsp104, its ability to dissociate preformed protofibrillar and fibrillar aggregates of Aβ was assessed in the presence and in the absence of ATP. Our results show that Hsp104 inhibits the fibrillization of monomeric and protofibrillar forms of Aβ in a concentration-dependent but ATP-independent manner. Inhibition of Aβ fibrillization by Hsp104 is observable up to Hsp104/Aβ stoichiometric ratios of 1:1000, suggesting a preferential interaction of Hsp104 with aggregation intermediates (e.g., oligomers, protofibrils, small fibrils) on the pathway of Aβ amyloid formation. This hypothesis is consistent with our observations that Hsp104 (i) interacts with Aβ protofibrils, (ii) inhibits conversion of protofibrils into amyloid fibrils, (iii) arrests fibril elongation and reassembly, and (iv) abolishes the capacity of protofibrils and sonicated fibrils to seed the fibrillization of monomeric Aβ. Together, these findings suggest that the strong inhibition of Aβ fibrillization by Hsp104 is mediated by its ability to act at different stages and target multiple intermediates on the pathway to amyloid formation.     

Interactions between Hsp70 and the hydrophobic core of alpha-synuclein inhibit fibril assembly

Molecular chaperones of the heat shock protein 70 (Hsp70) family counteract protein misfolding in a variety of neurodegenerative disease models. To determine whether human Hsp70 exerts similar effects on the aggregation of alpha-synuclein (alpha-Syn), the key component of insoluble fibrils present in Parkinson's disease, we investigated alpha-Syn fibril assembly in the presence of Hsp70. We found in vitro assembly was efficiently inhibited by substoichiometric concentrations of purified Hsp70 in the absence of cofactors. Experiments using alpha-Syn deletion mutants indicated that interactions between the Hsp70 substrate binding domain and the alpha-Syn core hydrophobic region underlie assembly inhibition. This assembly process was inhibited prior to the elongation stage as we failed to detect any fibrils by electron microscopy. In addition, fluorescence polarization and binding assays suggest that Hsp70 recognizes soluble alpha-Syn species in a highly dynamic and reversible manner. Together, these results provide novel insights into how Hsp70 suppresses alpha-Syn aggregation. Furthermore, our findings suggest that this critical step in Parkinson's disease pathogenesis may be subject to modulation by a common molecular chaperone.     

Pseudophosphorylation of tau protein directly modulates its aggregation kinetics

Hyperphosphorylation of tau protein is associated with neurofibrillary lesion formation in Alzheimer's disease and other tauopathic neurodegenerative diseases. It fosters lesion formation by increasing the concentration of free tau available for aggregation and by directly modulating the tau aggregation reaction. To clarify how negative charge incorporation into tau directly affects aggregation behavior, the fibrillization of pseudophosphorylation mutant T212E prepared in a full-length four-repeat tau background was examined in vitro as a function of time and submicromolar tau concentrations using electron microscopy assay methods. Kinetic constants for nucleation and extension phases of aggregation were then estimated by direct measurement and mathematical simulation. Kinetic analysis revealed that pseudophosphorylation increased tau aggregation rate by increasing the rate of filament nucleation. In addition, it increased aggregation propensity by stabilizing mature filaments against disaggregation. The data suggest that incorporation of negative charge into the T212 site can directly promote tau filament formation at multiple steps in the aggregation pathway.     

Design, synthesis, and biophysical properties of a helical Abeta1-42 analog: Inhibition of fibrillogenesis and cytotoxicity

The aggregation of amyloid β-peptide (Aβ) into β-sheet-rich aggregates is a crucial step in the etiology of Alzheimer’s disease. Helical forms of Aβ have been suggested to be intermediates in the aggregation process of the peptide in aqueous phase, micelles and membranes. A stable helical Aβ analog would be useful to investigate the role of helical intermediates in fibrillization by Aβ. Here we designed a helical analog by simply cross-linking the Cys residues of A30C, G37C-Aβ1-42 with 1,6-bismaleimidohexane. The analog assumed a weak α-helical conformation in model membranes mimicking lipid raft microdomains of neuronal membranes under conditions in which the wild-type Aβ1-42 formed a β-sheet, indicating the cross-linking locally induced a helical conformation. Furthermore, addition of equimolar helical Aβ analog significantly reduced the amyloid formation and cytotoxicity by Aβ1-42. Thus, our helical Aβ1-42 is not only a model peptide to investigate the role of helical intermediates in fibrillization by Aβ, but also an inhibitor of Aβ-induced cytotoxicity.     

Nucleation-dependent tau filament formation: the importance of dimerization and an estimation of elementary rate constants

Filamentous inclusions composed of the microtubule-associated protein tau are found in Alzheimer disease and other tauopathic neurodegenerative diseases, but the mechanisms underlying their formation from full-length protein monomer under physiological conditions are unclear. To address this issue, the fibrillization of recombinant full-length four-repeat human tau was examined in vitro as a function of time and submicromolar tau concentrations using electron microscopy assay methods and a small-molecule inducer of aggregation, thiazine red. Data were then fit to a simple homogeneous nucleation model with rate constant constraints established from filament dissociation rate, critical concentration, and mass-per-unit length measurements. The model was then tested by comparing the predicted time-dependent evolution of length distributions to experimental data. Results indicated that once assembly-competent conformations were attained, the rate-limiting step in the fibrillization pathway was tau dimer formation. Filament elongation then proceeded by addition of tau monomers to nascent filament ends. Filaments isolated at reaction plateau contained approximately 2 tau protomers/beta-strand spacing on the basis of mass-per-unit length measurements. The model suggests four key steps in the aggregation pathway that must be surmounted for tau filaments to form in disease.     

New class of inhibitors of amyloid-beta fibril formation. Implications for the mechanism of pathogenesis in Alzheimer's disease

The amyloid hypothesis suggests that the process of amyloid-beta protein (Abeta) fibrillogenesis is responsible for triggering a cascade of physiological events that contribute directly to the initiation and progression of Alzheimer's disease. Consequently, preventing this process might provide a viable therapeutic strategy for slowing and/or preventing the progression of this devastating disease. A promising strategy to achieve prevention of this disease is to discover compounds that inhibit Abeta polymerization and deposition. Herein, we describe a new class of small molecules that inhibit Abeta aggregation, which is based on the chemical structure of apomorphine. These molecules were found to interfere with Abeta1-40 fibrillization as determined by transmission electron microscopy, Thioflavin T fluorescence and velocity sedimentation analytical ultracentrifugation studies. Using electron microscopy, time-dependent studies demonstrate that apomorphine and its derivatives promote the oligomerization of Abeta but inhibit its fibrillization. Preliminary structural activity studies demonstrate that the 10,11-dihydroxy substitutions of the D-ring of apomorphine are required for the inhibitory effectiveness of these aporphines, and methylation of these hydroxyl groups reduces their inhibitory potency. The ability of these small molecules to inhibit Abeta amyloid fibril formation appears to be linked to their tendency to undergo rapid autoxidation, suggesting that autoxidation product(s) acts directly or indirectly on Abeta and inhibits its fibrillization. The inhibitory properties of the compounds presented suggest a new class of small molecules that could serve as a scaffold for the design of more efficient inhibitors of Abeta amyloidogenesis in vivo.     

alpha-Synuclein membrane interactions and lipid specificity

With the discovery of missense mutations (A53T and A30P) in alpha-synuclein (alpha-Syn) in several families with early onset familial Parkinson's disease, alpha-Syn aggregation and fibril formation have been thought to play a role in the pathogenesis of alpha-synucleinopathies, such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. As previous reports have suggested that alpha-Syn plays a role in lipid transport and synaptic membrane biogenesis, we investigated whether alpha-Syn binds to a specific lipid ligand using thin layer chromatography overlay and examined the changes in its secondary structure using circular dichroism spectroscopy. alpha-Syn was found to bind to acidic phospholipid vesicles and this binding was significantly augmented by the presence of phosphatidylethanolamine, a neutral phospholipid. We further examined the interaction of alpha-Syn with lipids by in situ atomic force microscopy. The association of soluble wild-type alpha-Syn with planar lipid bilayers resulted in extensive bilayer disruption and the formation of amorphous aggregates and small fibrils. The A53T mutant alpha-Syn disrupted the lipid bilayers in a similar fashion but at a slower rate. These results suggest that alpha-Syn membrane interactions are physiologically important and the lipid composition of the cellular membranes may affect these interactions in vivo.     

Secondary structural formation of alpha-synuclein amyloids as revealed by g-factor of solid-state circular dichroism

Alpha-synuclein (alpha-Syn) has been identified as a component of intracellular fibrillar deposits in Parkinson's disease. Though the real pathogenesis is still unknown, many investigations have revealed that conformational alteration and fibril formation of alpha-Syn protein have an important role in causing the disease. In this work, we introduced the g-factor spectra of solid-state circular dichroism to estimate the secondary structure contents of alpha-Syn fragments in amyloids. Fourier-transform infrared (FTIR) was also applied to confirm the structural formation. The results suggest that the central hydrophobic region is critical for beta-sheet formation and the conformational alteration is the foundation of protein abnormal aggregation. The research provides a practical approach to estimate the secondary structure contents of protein amyloids and further insight into the relevance of structural transformation and amyloidogenesis.     

Ligand-dependent inhibition and reversal of tau filament formation

Alzheimer's disease is defined in part by the intraneuronal accumulation of filaments comprised of the microtubule associated protein tau. Because animal model studies suggest that a toxic gain of function accompanies tau aggregation in neurons, selective pharmacological inhibitors of the process may have utility in slowing neurodegeneration. Here, the properties of a candidate small molecule inhibitor of tau fibrillization, 3-(2-hydroxyethyl)-2-[2-[[3-(2-hydroxyethyl)-5-methoxy-2-benzothiazolylidene]methyl]-1-butenyl]-5-methoxybenzothiazolium (N744), were characterized in vitro using transmission electron microscopy. N744 inhibited arachidonic acid-induced aggregation of full-length, four-repeat tau protein at substoichiometric concentrations relative to total tau and with an IC(50) of approximately 300 nM. Inhibition was accompanied by a dose-dependent decrease in the number concentration of filaments, suggesting that N744 interfered with tau filament nucleation. Stoichiometric concentrations of N744 also promoted tau disaggregation when added to mature synthetic filaments. Disaggregation followed first-order kinetics and was accompanied by a steady decrease in filament number, suggesting that N744 promoted endwise loss of tau molecules with limited filament breakage. N744 at substoichiometric concentrations did not inhibit Abeta and alpha-synuclein aggregation, indicating it was tau selective under these conditions. Because of its activity in vitro, N744 may offer a pharmacological approach to the role of tau fibrillization in neurodegeneration.     

The early events of alpha-synuclein oligomerization revealed by photo-induced cross-linking

Assembly of alpha-synuclein (alpha-Syn) into neurotoxic oligomers and fibrils is an important pathogenic feature of Parkinson's disease. Studying the early events of alpha-Syn aggregation, such as oligomerization and nucleation, is indispensable to understanding the complicated process. Here, photo-induced cross-linking of unmodified proteins (PICUP) technique is applied to elucidate the early-stage oligomerization of alpha-Syn. Results show that alpha-Syn in solution exhibits a mixture of various species, including at least monomers, dimers and trimers. Aggregation of alpha-Syn probably originates from the dimeric and trimeric seeds. Furthermore, the N-terminal amphipathic region is proposed to be required for the oligomerization (dimerization and trimerization) process. This observation may extend our knowledge on the early events of alpha-Syn aggregation and the neurotoxic aggregation species.     

Pseudophosphorylation and glycation of tau protein enhance but do not trigger fibrillization in vitro

Alzheimer's disease is defined in part by the intraneuronal aggregation of tau protein into filamentous lesions. The pathway is accompanied by posttranslational modifications including phosphorylation and glycation, each of which has been shown to promote tau fibrillization in vitro when present at high stoichiometry. To clarify the site-specific impact of posttranslational modification on tau fibrillization, the ability of recombinant full-length four repeat tau protein (htau40) and 11 pseudophosphorylation mutants to fibrillize in the presence of anionic inducer was assayed in vitro using transmission electron microscopy and laser light scattering assays. Tau glycated with d-glucose was examined as well. Both glycated tau and pseudophosphorylation mutants S199E, T212E, S214E, double mutant T212E/S214E, and triple mutant S199E/S202E/T205E yielded increased filament mass at equilibrium relative to wild-type tau. Increases in filament mass correlated strongly with decreases in critical concentration, indicating that both pseudophosphorylation and glycation promoted fibrillization by shifting equilibrium toward the fibrillized state. Analysis of reaction time courses further revealed that increases in filament mass were not associated with reduced lag times, indicating that these posttranslational modifications did not promote filament nucleation. The results suggest that site-specific posttranslational modifications can stabilize filaments once they nucleate, and thereby support their accumulation at low intracellular tau concentrations.     

Evidence for an intermediate in tau filament formation

Alzheimer's disease is defined in part by the intraneuronal accumulation of filaments comprised of the microtubule-associated protein tau. In vitro, fibrillization of full-length, unphosphorylated recombinant tau can be induced under near-physiological conditions by treatment with various agents, including anionic surfactants. Here we examine the pathway through which anionic surfactants promote tau fibrillization using a combination of electron microscopy and fluorescence spectroscopy. Protein and surfactant first interacted in solution to form micelles, which then provided negatively charged surfaces that accumulated tau aggregates. Surface aggregation of tau protein was followed by the time-dependent appearance of a thioflavin S reactive intermediate that accumulated over a period of hours. The intermediate was unstable in the absence of anionic surfaces, suggesting it was not filamentous. Fibrillization proceeded after intermediate formation with classic nucleation-dependent kinetics, consisting of lag phase followed by the exponential increase in filament lengths, followed by an equilibrium phase reached in approximately 24 h. The pathway did not require protein insertion into the micelle hydrophobic core or conformational change arising from mixed micelle formation, because anionic microspheres constructed from impermeable polystyrene were capable of qualitatively reproducing all aspects of the fibrillization reaction. It is proposed that the progression from amorphous aggregation through intermediate formation and fibrillization may underlie the activity of other inducers such as hyperphosphorylation and may be operative in vivo.     

Methylene blue inhibits amyloid Abeta oligomerization by promoting fibrillization

Amyloid plaques are hallmark neuropathological lesions in Alzheimer's disease, which consist of abnormally aggregated Abeta protein. Multiple Abeta aggregated species have been identified, and neurotoxicity appears to be correlated with the amount of nonfibrillar oligomers. Therefore, selective inhibition of Abeta oligomer formation has emerged as an attractive means of therapeutic intervention. To investigate whether small molecules can modulate aggregation to achieve selective inhibition of neurotoxic amyloid oligomers, Abeta aggregation was assayed in vitro in the presence of methylene blue, using immunoreactivity with the prefibrillar oligomer-specific antibody A11, transmission electron microscopy, and turbidity assays. Methylene blue inhibited oligomerization when used at substoichiometric concentrations relative to that of the Abeta monomer. Inhibition of Abeta oligomerization was achieved concomitant with promotion of fibrillization, suggesting that oligomer and fibril formation are distinct and competing pathways. Methylene blue-mediated promotion of fiber formation occurred via a dose-dependent decrease in the lag time and an increase in the fibrillization rate, consistent with promotion of both filament nucleation and elongation. Addition of methylene blue to preformed oligomers resulted in oligomer loss and promotion of fibrillization. The data show that Abeta oligomer formation is inhibited by promoting fibril formation, which suggests that the relative pathological significance of oligomers and fibrils may be tested in vivo using methylene blue. If Abeta oligomers represent the primary pathogenic species, then inhibition of this highly toxic species via promotion of formation of less toxic aggregates may be therapeutically useful.     

Potency of a tau fibrillization inhibitor is influenced by its aggregation state

Tau fibrillization is a potential therapeutic target for Alzheimer's and other neurodegenerative diseases. Several small-molecule inhibitors of tau aggregation have been developed for this purpose. One of them, 3,3'-bis(beta-hydroxyethyl)-9-ethyl-5,5'-dimethoxythiacarbocyanine iodide (N744), is a cationic thiacarbocyanine dye that inhibits recombinant tau filament formation when present at submicromolar concentrations. To prepare dosing regimens for testing N744 activity in biological models, its full concentration-effect relationship in the range 0.01-60muM was examined in vitro by electron microscopy and laser light scattering methods. Results revealed that N744 concentration dependence was biphasic, with fibrillization inhibitory activity appearing at submicromolar concentration, but with relief of inhibition and increases in fibrillization apparent above 10muM. Therefore, fibrillization was inhibited 50% only over a narrow concentration range, which was further reduced by filament stabilizing modifications such as tau pseudophosphorylation. N744 inhibitory activity also was paralleled by changes in its aggregation state, with dimer predominating at inhibitory concentrations and large dye aggregates appearing at high concentrations. Ligand dimerization was promoted by the presence of tau protein, which lowered the equilibrium dissociation constant for dimerization more than an order of magnitude relative to controls. The results suggest that ligand aggregation may play an important role in both inhibitory and disinhibitory phases of the concentration-effect curve, and may lead to complex dose-response relationships in model systems.     

The Role of Pro, Gly Lys, and Arg Containing Peptides on Amyloid-Beta Aggregation

Genetic, biochemical and pathological evidence support that self-assembly of amyloid-beta (Aβ) peptide into toxic aggregates is implicated as the cause of Alzheimer’s disease. An attractive therapeutic strategy for the treatment of AD is to prevent or interfere with Aβ aggregation. A systematic investigation of the effects of proline-, glycine-, arginine- and lysine- containing peptides (PGKLVYA, KKLVFFARRRRA and KKLVFFA) on the beta-amyloid aggregation was made using FTIR, circular dichroism, ANS binding, ThT binding and TEM analysis. These peptides are based on the central hydrophobic region of Aβ (residues 16–20), which is believed to be crucial in Aβ self-association. There is increasing evidence to suggest that protein aggregation, including amyloid fibril formation results from the strong self-association tendency of the partially folded intermediates. Addition of PGKLVYA and KKLVFFARRRRA resulted in increase in ANS fluorescence intensity, suggesting enhanced exposure of hydrophobic surface area. As observed by ThT and TEM analysis PGKLVYA and KKLVFFARRRRA promote non-fibrillar ensembles, while peptide KKLVFFA accelerated the fibrillization of Aβ peptide by stabilizing intermolecular interactions. Circular dichroism and FTIR data showed that PGKLVYA and KKLVFFARRRRA effectively prevented amyloid-beta (Aβ) peptide adopting the beta-sheet secondary structure correlated with fibrillogenesis. This result indicates that PGKLVYA and KKLVFFARRRRA might have triggered another mechanism of Aβ assembly.     

Controlling aggregation propensity in A53T mutant of alpha-synuclein causing Parkinson’s disease

Understanding α-synuclein in terms of fibrillization, aggregation, solubility and stability is fundamental in Parkinson’s disease (PD). The three familial mutations, namely, A30P, E46K and A53T cause PD because the hydrophobic regions in α-synuclein acquire β-sheet configuration, and have a propensity to fibrillize and form amyloids that cause cytotoxicity and neurodegeneration. On simulating the native form and mutants (A30P, E46K and A53T) of α-synuclein in water solvent, clear deviations are observed in comparison to the all-helical 1XQ8 PDB structure. We have identified two crucial residues, ⁴⁰Val and ⁷⁴Val, which play key roles in β-sheet aggregation in the hydrophobic regions 36-41 and 68-78, respectively, leading to fibrillization and amyloidosis in familial (A53T) PD. We have also identified V40D_V74D, a double mutant of A53T (the most amyloidogenic mutant). The simultaneous introduction of these two mutations in A53T nearly ends its aggregation propensity, increases its solubility and positively enhances its thermodynamic stability.