The genetic missense A30P mutation of the wild-type α-synuclein
protein results in the replacement of the 30th amino acid residue
from alanine (Ala) to proline (Pro) and was initially found in the
members of a German family who developed Parkinson’s disease.
Even though the structures of these proteins have been measured before,
detailed understanding about the structures and their relationships
with free energy landscapes is lacking, which is of interest to provide
insights into the pathogenic mechanism of Parkinson’s disease.
We report the secondary and tertiary structures and conformational
free energy landscapes of the wild-type and A30P mutant-type α-synuclein
proteins in an aqueous solution environment via extensive parallel
tempering molecular dynamics simulations along with thermodynamic
calculations. In addition, we present the residual secondary structure
component transition stabilities at the atomic level with dynamics
in terms of free energy change calculations using a new strategy that
we reported most recently. Our studies yield new interesting results;
for instance, we find that the A30P mutation has local as well as
long-range effects on the structural properties of the wild-type α-synuclein
protein. The helical content at Ala18-Gly31 is less prominent in comparison
to the wild-type α-synuclein protein. The β-sheet structure
abundance decreases in the N-terminal region upon A30P mutation of
the wild-type α-synuclein, whereas the NAC and C-terminal regions
possess larger tendencies for β-sheet structure formation. Long-range
intramolecular protein interactions are less abundant upon A30P mutation,
especially between the NAC and C-terminal regions, which is linked
to the less compact and less stable structures of the A30P mutant-type
rather than the wild-type α-synuclein protein. Results including
the usage of our new strategy for secondary structure transition stabilities
show that the A30P mutant-type α-synuclein tendency toward aggregation
is higher than the wild-type α-synuclein but we also find that
the C-terminal and NAC regions of the A30P mutant-type α-synuclein
are reactive toward fibrillzation and aggregation based on atomic
level studies with dynamics in an aqueous solution environment. Therefore,
we propose that small molecules or drugs blocking the specific residues,
which we report herein, located in the NAC- and C-terminal regions
of the A30P mutant-type α-synuclein protein might help to reduce
the toxicity of the A30P mutant-type α-synuclein protein.
α-Synuclein; genetic missense mutation; free energy landscape; molecular dynamics simulations
α-Synuclein is the major component of pathological inclusions characteristic of diseases like Parkinson’s disease, dementia with Lewy bodies, and multiple systems atrophy. A role for α-synuclein in neurodegenerative diseases is further supported by point mutations and duplication/triplication of the α-synuclein gene (SNCA) that are causative of these disorders. The middle hydrophobic region of the α-synuclein protein, also termed the “non-Aβ component of Alzheimer’s disease amyloid plaque (NAC)” domain, is required for α-synuclein to polymerize into amyloid filaments, which are the major components of α-synuclein pathological inclusions. In the current studies, we assessed the importance of specific stretches of hydrophobic residues in driving the intrinsic ability of α-synuclein to polymerize. Several small deletions, even one as short as 2 amino acid residues (A76 and V77), dramatically impaired the ability of α-synuclein to polymerize into mature amyloidogenic fibrils, and, instead, preferentially formed oligomers. However, this inhibition of filament assembly was clearly dependent on the spatial context, since similar and larger hydrophobic deletions in other parts of the NAC domain only reduced the rate of fibril formation, without abrogating filament assembly. Further, mutation of residue E83 to an A rescued the ability of mutant Δ76–77 α-synuclein to polymerize. These findings support the notion that while both the location and hydrophobicity of protein segments are important elements that affect the propensity to form amyloid fibrils, the intrinsic ability of a polypeptide to structural fold into amyloid is also critical.
Parkinson's disease (PD) involves the selective damage of dopaminergic neuron cells resulting from the accumulation and fibril formation of alpha-synuclein. Recently, it has been shown that not only full-length alpha-synuclein, but also C-terminal truncated forms exist in the normal brain, as well as Lewy bodies, which are cytoplasmic inclusions in PD. It is known that truncated alpha-synuclein has a much higher ability to aggregate and fibrillate than full-length alpha-synuclein. Since the fibrils and precursor oligomers of alpha-synuclein are cytotoxic to the neuron, inhibitors that prevent the formation of oligomers and/or fibrils might open the way to a novel therapeutic approach to PD. However, no inhibitor for truncated alpha-synuclein has been reported yet.
In this study, we first characterized the aggregation and cytotoxicity of C-truncated alpha-synuclein119 and alpha-synuclein133 which have been found in both the normal and the pathogenic brain. Alpha-synuclein119 aggregated more rapidly and enhanced significantly the fibril formation of alpha-synuclein. Although both of alpha-synuclein119 and alpha-synuclein133 showed a high cytotoxicity, alpha-synuclein133 showed a similar aggregation with full-length alpha-synuclein and no acceleration effect. We showed that PQQ dramatically inhibits the fibril formation of C-terminal truncated alpha-synuclein110119, and 133 as well as the mixtures of full-length alpha-synuclein with these truncated variants. Moreover, PQQ decreases the cytotoxicity of truncated alpha-synuclein.
Our results demonstrate that PQQ inhibits the amyloid fibril formation and cytotoxicity of the C-truncated alpha-synuclein variants. We believe that PQQ is a strong candidate for a reagent compound in the treatment of PD.
Aggregation of α-synuclein (αSyn), the primary protein component in Lewy body inclusions of patients with Parkinson’s disease, arises when the normally soluble intrinsically disordered protein converts to amyloid fibrils. In this work, we provide a mechanistic view of the role of N-terminal acetylation on fibrillation by first establishing a quantitative relationship between monomer secondary structural propensity and fibril assembly kinetics, and secondly by demonstrating in the N-terminal acetylated form of the early onset A53T mutation, that N-terminal transient helices formed and/or inhibited by N-terminal acetylation modulate the fibril assembly rates. Using NMR chemical shifts and fluorescence experiments, we report that secondary structural propensity in residues 5–8, 14–31, and 50–57 are highly correlated to fibril growth rate. A four-way comparison of secondary structure propensity and fibril growth rates of N-terminally acetylated A53T and WT αSyn with non-acetylated A53T and WT αSyn present novel mechanistic insight into the role of N-terminal acetylation in amyloid fibril formation. We show that N-terminal acetylation inhibits the formation of the “fibrillation promoting” transient helix at residues 14–31 resulting from the A53T mutation in the non-acetylated variant and supports the formation of the “fibrillation inhibiting” transient helix in residues 1–12 thereby resulting in slower fibrillation rates relative to the previously studied non-acetylated A53T variant. Our results highlight the critical interplay of the region-specific transient secondary structure of the N-terminal region with fibrillation, and the inhibitory role of the N-terminal acetyl group in fibril formation.
The E46K genetic missense mutation of the wild-type α-synuclein
protein was recently identified in a family of Spanish origin with
hereditary Parkinson’s disease. Detailed understanding of the
structures of the monomeric E46K mutant-type α-synuclein protein
as well as the impact of the E46K missense mutation on the conformations
and free energy landscapes of the wild-type α-synuclein are
required for gaining insights into the pathogenic mechanism of Parkinson’s
disease. In this study, we use extensive parallel tempering molecular
dynamics simulations along with thermodynamic calculations to assess
the secondary and tertiary structural properties as well as the conformational
preferences of the monomeric wild-type and E46K mutant-type α-synuclein
proteins in an aqueous solution environment. We also present the residual
secondary structure component conversion stabilities with dynamics
using a theoretical strategy, which we most recently developed. To
the best of our knowledge, this study presents the first detailed
comparison of the structural and thermodynamic properties of the wild-type
and E46K mutant-type α-synuclein proteins in an aqueous solution
environment at the atomic level with dynamics. We find that the E46K
mutation results not only in local but also in long-range changes
in the structural properties of the wild-type α-synuclein protein.
The mutation site shows a significant decrease in helical content
as well as a large increase in β-sheet structure formation upon
E46K mutation. In addition, the β-sheet content of the C-terminal
region increases significantly in the E46K mutant-type αS in
comparison to the wild-type αS. Our theoretical strategy developed
to assess the thermodynamic preference of secondary structure transitions
indicates that this shift in secondary structure is the result of
a decrease in the thermodynamic preference of turn to helix conversions
while the coil to β-sheet preference increases for these residues.
Long-range intramolecular protein interactions of the C-terminal with
the N-terminal and NAC regions increase upon E46K mutation, resulting
in more compact structures for the E46K mutant-type rather than wild-type
αS. However, the E46K mutant-type αS structures are less
stable than the wild-type αS. Overall, our results show that
the E46K mutant-type αS has a higher propensity to aggregate
than the wild-type αS and that the N-terminal and C-terminal
regions are reactive toward fibrillization and aggregation upon E46K
mutation and we explain the associated reasons based on the structural
properties herein. Small molecules or drugs that can block the specific
residues forming abundant β-sheet structure, which we report
here, might help to reduce the reactivity of these intrinsically disordered
fibrillogenic proteins toward aggregation and their toxicity.
α-synuclein; genetic missense mutation; free energy landsape; molecular dynamics
α-Synuclein is an abundant presynaptic protein that binds to phospholipids and synaptic vesicles. Physiologically, α-synuclein functions as a SNARE-protein chaperone that promotes SNARE-complex assembly for neurotransmitter release. Pathologically, α-synuclein mutations and α-synuclein overexpression cause Parkinson’s disease, and aggregates of α-synuclein are found as Lewy bodies in multiple neurodegenerative disorders (“synucleinopathies”). The relation of the physiological functions and pathological effects of α-synuclein remain unclear. As an initial avenue of addressing this question, we here systematically examined the effect of α-synuclein mutations on its physiological and pathological activities. We generated 26 α-synuclein mutants spanning the entire molecule, and analyzed them in comparison to wild-type α-synuclein in seven assays that range from biochemical studies with purified α-synuclein to expression of α-synuclein in cultured neurons to examination of the effects of virally expressed α-synuclein that was introduced into the mouse substrantia nigra by stereotactic injections. We found that both the N- and C-terminal sequences of α-synuclein were required for its physiological function as SNARE-complex chaperone, but that these sequences were not essential for its neuropathological effects. In contrast, point mutations in the region of α-synuclein referred to as non-amyloid β component (NAC; residues 61–95) as well as point mutations linked to Parkinson’s disease (A30P, E46K, and A53T) increased the neurotoxicity of α-synuclein but did not affect its physiological function. Thus, our data show that the physiological function of α-synuclein, although protective of neurodegeneration in some contexts, is fundamentally distinct from its neuropathological effects, thereby dissociating the two activities of α-synuclein.
The toxicity of α-synuclein in vivo is not well understood. Rockenstein et al. describe an α-synuclein transgenic model expressing the E57K mutant that forms stable oligomers. They show that oligomers accumulate at synapses and that the mutation interferes with synaptic vesicles and is associated with behavioural deficits and neurodegeneration.
In Parkinson’s disease and dementia with Lewy bodies, α-synuclein aggregates to form oligomers and fibrils; however, the precise nature of the toxic α-synuclein species remains unclear. A number of synthetic α-synuclein mutations were recently created (E57K and E35K) that produce species of α-synuclein that preferentially form oligomers and increase α-synuclein-mediated toxicity. We have shown that acute lentiviral expression of α-synuclein E57K leads to the degeneration of dopaminergic neurons; however, the effects of chronic expression of oligomer-prone α-synuclein in synapses throughout the brain have not been investigated. Such a study could provide insight into the possible mechanism(s) through which accumulation of α-synuclein oligomers in the synapse leads to neurodegeneration. For this purpose, we compared the patterns of neurodegeneration and synaptic damage between a newly generated mThy-1 α-synuclein E57K transgenic mouse model that is prone to forming oligomers and the mThy-1 α-synuclein wild-type mouse model (Line 61), which accumulates various forms of α-synuclein. Three lines of α-synuclein E57K (Lines 9, 16 and 54) were generated and compared with the wild-type. The α-synuclein E57K Lines 9 and 16 were higher expressings of α-synuclein, similar to α-synuclein wild-type Line 61, and Line 54 was a low expressing of α-synuclein compared to Line 61. By immunoblot analysis, the higher-expressing α-synuclein E57K transgenic mice showed abundant oligomeric, but not fibrillar, α-synuclein whereas lower-expressing mice accumulated monomeric α-synuclein. Monomers, oligomers, and fibrils were present in α-synuclein wild-type Line 61. Immunohistochemical and ultrastructural analyses demonstrated that α-synuclein accumulated in the synapses but not in the neuronal cells bodies, which was different from the α-synuclein wild-type Line 61, which accumulates α-synuclein in the soma. Compared to non-transgenic and lower-expressing mice, the higher-expressing α-synuclein E57K mice displayed synaptic and dendritic loss, reduced levels of synapsin 1 and synaptic vesicles, and behavioural deficits. Similar alterations, but to a lesser extent, were seen in the α-synuclein wild-type mice. Moreover, although the oligomer-prone α-synuclein mice displayed neurodegeneration in the frontal cortex and hippocampus, the α-synuclein wild-type only displayed neuronal loss in the hippocampus. These results support the hypothesis that accumulating oligomeric α-synuclein may mediate early synaptic pathology in Parkinson’s disease and dementia with Lewy bodies by disrupting synaptic vesicles. This oligomer-prone model might be useful for evaluating therapies directed at oligomer reduction.
α-synuclein; transgenic; oligomer; Parkinson’s disease; synaptic vesicles
Indolic derivatives can affect fibril growth of amyloid forming proteins. The neurotransmitter serotonin (5-HT) is of particular interest, as it is an endogenous molecule with a possible link to neuropsychiatric symptoms of Parkinson disease. A key pathomolecular mechanism of Parkinson disease is the misfolding and aggregation of the intrinsically unstructured protein α-synuclein. We performed a biophysical study to investigate an influence between these two molecules. In an isolated in vitro system, 5-HT interfered with α-synuclein amyloid fiber maturation, resulting in the formation of partially structured, SDS-resistant intermediate aggregates. The C-terminal region of α-synuclein was essential for this interaction, which was driven mainly by electrostatic forces. 5-HT did not bind directly to monomeric α-synuclein molecules and we propose a model where 5-HT interacts with early intermediates of α-synuclein amyloidogenesis, which disfavors their further conversion into amyloid fibrils.
► The neurotransmitter serotonin (5-HT) suppresses amyloid fibril growth of alpha-synuclein (AS). ► 5-HT binds to intermediate aggregates of alpha-synuclein, not to monomeric AS. Consequently, 5-HT does not influence initial steps of amyloidogenesis. ► 5-HT promotes the accumulation of partially structured, SDS-resistant “on pathway” aggregates of AS. ► The C-terminal region of AS is essential for a charge dependent interaction. ► “On pathway” and “off-pathway” aggregations of AS might mechanistically overlap.
AS, α-synuclein; 5-HT, serotonin; 5,7-HT, 5,7 dihydroxytryptamine; 5-HIAA, 5-hydroxyindoleacetic acid; ThioT, thioflavin T; TEM, transmission electron spectroscopy; DLS, dynamic light scattering; NAC-region, non-Aβ component region; Protein misfolding; Amyloid; Aggregation; Parkinson disease; Neurodegeneration; Indoleamine
α-synuclein is the major component of filamentous Lewy bodies found in the brains of patients diagnosed with Parkinson’s disease. Recent studies demonstrate that, in addition to the wild-type sequence, α-synuclein is found in several modified forms, including truncated and phosphorylated species. Although the mechanism by which the neuronal loss in Parkinson’s disease occurs is unknown, aggregation and fibril formation of α-synuclein is considered to be a key pathological feature. In this study we analyze the rates of fibril formation and the monomer-fibril equilibrium for eight disease-associated truncated and phosphorylated α-synuclein variants. Comparison of the relative rates of aggregation reveals a strong monotonic relationship between the C-terminal charge of α-synuclein and the lag time prior to the observation of fibril formation, with truncated species exhibiting the fastest aggregation rates. Moreover, we find that a decrease in C-terminal charge shifts the equilibrium to favor the fibrillar species. An analysis of these findings in the context of linear growth theories suggests that the loss of the charge-mediated stabilization of the soluble state is responsible for the enhanced aggregation rate and increased extent of fibril fraction. Therefore, C-terminal charge is kinetically and thermodynamically protective against α-synuclein polymerization and may provide a target for the treatment of Parkinson’s Disease.
Aromatic-aromatic and aromatic-hydrophobic interactions have been proposed to play a role in amyloid formation by a range of polypeptides including islet amyloid polypeptide (IAPP, Amylin). IAPP is responsible for amyloid formation during type-2 diabetes. The polypeptide is 37 residues in length and contains three aromatic residues Phe-15, Phe-23, and Tyr-37. The ability of all single aromatic to leucine mutants, all double aromatic to leucine mutants and the triple leucine mutant to form amyloid were examined. Amyloid formation was almost twice as rapid for the F15L mutant relative to wild-type, but was almost three fold slower for the Y37L mutant and almost two fold slower for F23L mutant. Amyloid fibrils formed from each of the single mutants were effective at seeding amyloid formation by wild-type IAPP, implying that the fibril structures are similar. The F15LF23L double mutant has a larger effect than the F15LY37L double mutant on the rate of amyloid formation, even though a Y37L substitution has more drastic consequences in the wild-type background than does the F23L mutation, suggesting non-additive effects between the different sites. The triple leucine mutant and the F23LY37L double mutant are the slowest to form amyloid. F15 has been proposed to make important contacts early in the aggregation pathway, but the F15L mutant data indicates that they are not optimal. A set of variants containing natural and unnatural amino acids at position 15, which were designed to conserve hydrophobicity, but which alter α-helix and β-sheet propensity, were analyzed to determine the properties of this position that control the rate of amyloid formation. There is no correlation between β-sheet propensity at this position and the rate of amyloid formation, but there is a correlation with α-helical propensity.
IAPP; Islet amyloid polypeptide; Amylin; Amyloid; Aromatic interactions; Pi-interactions
Merozoite surface protein 2 (MSP2) from the human malaria parasite Plasmodium falciparum is expressed as a GPI-anchored protein on the merozoite surface. MSP2 is assumed to have a role in erythrocyte invasion and is a leading vaccine candidate. Recombinant MSP2 forms amyloid-like fibrils upon storage, as do peptides corresponding to sequences in the conserved N-terminal region, which constitutes the structural core of fibrils formed by full-length MSP2. We have investigated the roles of individual residues in fibril formation and local ordered structure in two peptides, a recombinant 25-residue peptide corresponding to the entire N-terminal domain of mature MSP2 and an 8-residue peptide from the central region of this domain (residues 8–15). Both peptides formed fibrils that were similar to amyloid-like fibrils formed by full-length MSP2. Phe11 and Ile12 have important roles both in stabilising local structure in these peptides and promoting fibril formation; the F11A and I12A mutants of MSP28–15 were essentially unstructured in solution and fibril formation at pH 7.4 and 4.7 was markedly retarded. The T10A mutant showed intermediate behaviour, having a less well-defined structure than wild-type and slower fibril formation at pH 7.4. The mutation of Phe11 and Ile12 in MSP21–25 significantly retarded but did not abolish fibril formation, indicating that these residues also play a key role in fibril formation by the entire N-terminal conserved region. These mutations had little effect on the aggregation of full-length MSP2, however, suggesting that regions outside the conserved N-terminus have unanticipated importance for fibril formation in the full-length protein.
malaria; fibril; amyloid; nuclear magnetic resonance; structure
Several studies have shown that catecholamines can inhibit the fibrillation of α-synuclein, 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 DOPAC (3, 4-dihydroxyphenylacetic acid), a normal product of the metabolism of dopamine, can inhibit the fibrillation of α-synuclein (α-Syn), due to non-covalent binding of DOPAC to α-Syn monomer. Intriguingly, the presence of α-synuclein 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 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-synuclein 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, catechol), their 1,4-dihydroxyphenyl isomers (hydroquinone, homogentisic acid) are able to modify α-Syn covalently, probably due to the less steric hindrance in the Michael addition.
α-synuclein; DOPAC; DOPAC-stabilized oligomer; amyloid fibril; oxidative modification; methionine oxidation; dopamine
Parkinson’s disease (PD) is a slowly progressive movement disorder that results from the loss of dopaminergic neurons in the substantia nigra, a small area of cells in the mid-brain. PD is a multifactorial disorder with unknown etiology, in which both genetic and environmental factors play important roles. Substantial evidence links α-synuclein, a small highly conserved presynaptic protein with unknown function, to both familial and sporadic PD. Rare familial cases of PD are associated with missense point mutations in α-synuclein, or with the hyper-expression of the wild type protein due to its gene duplication/triplication. Furthermore, α-synuclein was identified as the major component of amyloid fibrils found in Lewy body and Lewy neurites, the characteristic proteinaceous deposits that are the diagnostic hallmarks of PD. α-Synuclein is abundant in various regions of the brain and has two closely related homologs, β-synuclein and γ-synuclein. When isolated in solution, the protein is intrinsically disordered, but in the presence of lipid surfaces α-synuclein adopts a highly helical structure that is believed to mediate its normal function(s). A number of different conformational states of α-synuclein have been observed. Besides the membrane-bound form, other critical conformations include a partially-folded state that is a key intermediate in aggregation and fibrillation, various oligomeric species, and fibrillar and amorphous aggregates. A number of intrinsic and extrinsic factors that either accelerate or inhibit the rate of α-synuclein aggregation and fibrillation in vitro are known. There is a strong correlation between the conformation of α-synuclein (induced by various factors) and its rate of fibrillation. The aggregation process appears to be branched, with one pathway leading to fibrils and another to oligomeric intermediates that may ultimately form amorphous deposits. The molecular basis of Parkinson’s disease appears to be tightly coupled to the aggregation of α-synuclein and the factors that affect its conformation. This review focuses on the contributions of Prof. Anthony L. Fink to the field and presents some recent developments in this exciting area.
α-Synuclein; synucleinopathies; aggregation; amyloid; fibril; neurodegeneration; intrinsically disordered protein; NMR; partially folded intermediate
Fibrillar αSynuclein is the major constituent of Lewy bodies and Lewy neurites, the protein deposits characteristic for Parkinson’s disease (PD). Multiplications of the αSynuclein gene, as well as point mutations cause familial PD. However, the exact role of αSynuclein in neurodegeneration remains uncertain. Recent research in invertebrates has suggested that oligomeric rather than fibrillizing αSynuclein mediates neurotoxicity. To investigate the impact of αSynuclein aggregation on the progression of neurodegeneration, we expressed variants with different fibrillation propensities in the rat substantia nigra (SN) by means of recombinant adeno-associated viral (AAV) vectors. The formation of proteinase K-resistant αSynuclein aggregates was correlated to the loss of nigral dopaminergic (DA) neurons and striatal fibers. Expression of two prefibrillar, structure-based design mutants of αSynuclein (i.e., A56P and A30P/A56P/A76P) resulted in less aggregate formation in nigral DA neurons as compared to human wild-type (WT) or the inherited A30P mutation. However, only the αSynuclein variants capable of forming fibrils (WT/A30P), but not the oligomeric αSynuclein species induced a sustained progressive loss of adult nigral DA neurons. These results demonstrate that divergent modes of αSynuclein neurotoxicity exist in invertebrate and mammalian DA neurons in vivo and suggest that fibrillation of αSynuclein promotes the progressive degeneration of nigral DA neurons as found in PD patients.
αSynuclein; Parkinson’s disease; Aggregation; Adeno-associated virus; Substantia nigra
α-Synuclein is the major pathological component of synucleinopathies including Parkinson's disease and dementia with Lewy bodies. Recent studies have demonstrated that α-synuclein also plays important roles in the release of synaptic vesicles and synaptic membrane recycling in healthy neurons. However, the precise relationship between the pathogenicity and physiological functions of α-synuclein remains to be elucidated. To address this issue, we investigated the subcellular localization of α-synuclein in normal and pathological conditions using primary mouse hippocampal neuronal cultures. While some neurons expressed high levels of α-synuclein in presynaptic boutons and cell bodies, other neurons either did not or only very weakly expressed the protein. These α-synuclein-negative cells were identified as inhibitory neurons by immunostaining with specific antibodies against glutamic acid decarboxylase (GAD), parvalbumin, and somatostatin. In contrast, α-synuclein-positive synapses were colocalized with the excitatory synapse marker vesicular glutamate transporter-1. This expression profile of α-synuclein was conserved in the hippocampus in vivo. In addition, we found that while presynaptic α-synuclein colocalizes with synapsin, a marker of presynaptic vesicles, it is not essential for activity-dependent membrane recycling induced by high potassium treatment. Exogenous supply of preformed fibrils generated by recombinant α-synuclein was shown to promote the formation of Lewy body (LB) -like intracellular aggregates involving endogenous α-synuclein. GAD-positive neurons did not form LB-like aggregates following treatment with preformed fibrils, however, exogenous expression of human α-synuclein allowed intracellular aggregate formation in these cells. These results suggest the presence of a different mechanism for regulation of the expression of α-synuclein between excitatory and inhibitory neurons. Furthermore, α-synuclein expression levels may determine the efficiency of intracellular aggregate formation in different neuronal subtypes.
The [Het-s] prion of the fungus Podospora anserina represents a good model system for studying the structure-function relationship in amyloid proteins because a high resolution solid-state NMR structure of the amyloid prion form of the HET-s prion forming domain (PFD) is available. The HET-s PFD adopts a specific β-solenoid fold with two rungs of β-strands delimiting a triangular hydrophobic core. A C-terminal loop folds back onto the rigid core region and forms a more dynamic semi-hydrophobic pocket extending the hydrophobic core. Herein, an alanine scanning mutagenesis of the HET-s PFD was conducted. Different structural elements identified in the prion fold such as the triangular hydrophobic core, the salt bridges, the asparagines ladders and the C-terminal loop were altered and the effect of these mutations on prion function, fibril structure and stability was assayed. Prion activity and structure were found to be very robust; only a few key mutations were able to corrupt structure and function. While some mutations strongly destabilize the fold, many substitutions in fact increase stability of the fold. This increase in structural stability did not influence prion formation propensity in vivo. However, if an Ala replacement did alter the structure of the core or did influence the shape of the denaturation curve, the corresponding variant showed a decreased prion efficacy. It is also the finding that in addition to the structural elements of the rigid core region, the aromatic residues in the C-terminal semi-hydrophobic pocket are critical for prion propagation. Mutations in the latter region either positively or negatively affected prion formation. We thus identify a region that modulates prion formation although it is not part of the rigid cross-β core, an observation that might be relevant to other amyloid models.
Prions are infectious protein particles causing fatal diseases in mammals. Prions correspond to self-perpetuating amyloid protein polymers. Prions also exist in fungi where they behave as cytoplasmic infectious elements. The [Het-s] prion of the fungus Podospora anserina constitutes a favorable model for the analysis of the structural basis of prion propagation because a high resolution structure of the prion form of [Het-s] is available, a situation so far unique to this prion model. We have analyzed the relation between [Het-s] structure and function using alanine scanning mutagenesis. We have generated 32 single amino acid variants of the prion forming domain and analyzed their prion function in vivo and structure by solid-state NMR. We find that the PFD structure is very robust and that only a few key mutations affect prion structure and function. In addition, we find that a C-terminal semi-flexible loop plays a critical role in prion propagation although it is not part of rigid amyloid core. This study offers insights on the structural basis of prion propagation and illustrates that accessory regions outside of the amyloid core can critically participate in prion function, an observation that could be relevant to other amyloid models.
Aggregated α-synuclein and the point mutations Ala30Pro and Ala53Thr of α-synuclein are associated with Parkinson’s disease. The physiological roles of α-synuclein and methionine oxidation of the α-synuclein protein structure and function are not fully understood. Methionine sulfoxide reductase A (MsrA) reduces methionine sulfoxide residues and functions as an antioxidant. To monitor the effect of methionine oxidation to α-synuclein on basic cellular processes, α-synucleins were expressed in msrA null mutant and wild-type yeast cells. Protein degradation was inhibited in the α-synuclein-expressing msrA null mutant cells compared to α-synuclein-expressing wild-type cells. Increased inhibition of degradation and elevated accumulations of fibrillated proteins were observed in SynA30P-expressing msrA null mutant cells. Additionally, methionine oxidation inhibited α-synuclein phosphorylation in yeast cells and in vitro by casein kinase 2. Thus, a compromised MsrA function combined with α-synuclein overexpression may promote processes leading to synucleinopathies.
Oxidative stress; Posttranslation modification; Neurodegenerative diseases; Parkinson’s disease; Antioxidants; Protein aggregation; Yeast; Synuclein
Fibrils derived from Pmel17 are functional amyloids upon which melanin is deposited. Fibrils of the repeat domain (RPT) from Pmel17 form under strict melanosomal pH (4.5–5.5) and completely dissolve at pH ≥ 6. To determine which Glu residue is responsible for this reversibility, aggregation of single, double, and quadruple Ala- and Gln-mutants were examined by intrinsic Trp fluorescence, circular dichroism spectroscopy, and transmission electron microscopy. Charge neutralization of either E404, E422, E425, or E430, glutamic acids located in the putative amyloid-forming region, modulated aggregation kinetics. Remarkably, the removal of a single negative charge at E422 out of a total of 16 carboxylic acids shifted the pH dependence by a full pH unit. Mutation at E404, E425, or E430 has little to no effect. We suggest that protonation at E422 is essential for initiating amyloid formation while other Glu residues play an allosteric role in fibril stability.
Pmel17; repeat domain; melanin; TEM; tryptophan
Membrane-induced disorder-to-helix transition of α-synuclein, a presynaptic protein, has been implicated in a number of important neuronal functions as well as in the etiology of Parkinson’s disease. In order to obtain structural insights of membrane-bound α-synuclein at the residue-specific resolution, we took advantage of the fact that the protein is devoid of tryptophan and incorporated single tryptophan at various residue positions along the sequence. These tryptophans were used as site-specific markers to characterize the structural and dynamical aspects of α-synuclein on the negatively charged small unilamellar lipid vesicles. An array of site-specific fluorescence readouts, such as the spectral-shift, quenching efficiency and anisotropy, allowed us to discern various features of the conformational rearrangements occurring at different locations of α-synuclein on the lipid membrane. In order to define the spatial localization of various regions of the protein near the membrane surface, we utilized a unique and sensitive indicator, namely, red-edge excitation shift (REES), which originates when a fluorophore is located in a highly ordered micro-environment. The extent of REES observed at different residue positions allowed us to directly identify the residues that are localized at the membrane-water interface comprising a thin (∼ 15 Å) layer of motionally restrained water molecules and enabled us to construct a dynamic hydration map of the protein. The combination of site-specific fluorescence readouts allowed us to unravel the intriguing molecular details of α-synuclein on the lipid membrane in a direct model-free fashion. Additionally, the combination of methodologies described here are capable of distinguishing subtle but important structural alterations of α-synuclein bound to different negatively charged lipids with varied head-group chemistry. We believe that the structural modulations of α-synuclein on the membrane could potentially be related to its physiological functions as well as to the onset of Parkinson’s diseases.
Human α-Synuclein (αSyn) is a natively unfolded protein whose aggregation into amyloid fibrils is involved in the pathology of Parkinson disease. A full comprehension of the structure and dynamics of early intermediates leading to the aggregated states is an unsolved problem of essential importance to researchers attempting to decipher the molecular mechanisms of αSyn aggregation and formation of fibrils. Traditional bulk techniques used so far to solve this problem point to a direct correlation between αSyn's unique conformational properties and its propensity to aggregate, but these techniques can only provide ensemble-averaged information for monomers and oligomers alike. They therefore cannot characterize the full complexity of the conformational equilibria that trigger the aggregation process. We applied atomic force microscopy–based single-molecule mechanical unfolding methodology to study the conformational equilibrium of human wild-type and mutant αSyn. The conformational heterogeneity of monomeric αSyn was characterized at the single-molecule level. Three main classes of conformations, including disordered and “β-like” structures, were directly observed and quantified without any interference from oligomeric soluble forms. The relative abundance of the “β-like” structures significantly increased in different conditions promoting the aggregation of αSyn: the presence of Cu2+, the pathogenic A30P mutation, and high ionic strength. This methodology can explore the full conformational space of a protein at the single-molecule level, detecting even poorly populated conformers and measuring their distribution in a variety of biologically important conditions. To the best of our knowledge, we present for the first time evidence of a conformational equilibrium that controls the population of a specific class of monomeric αSyn conformers, positively correlated with conditions known to promote the formation of aggregates. A new tool is thus made available to test directly the influence of mutations and pharmacological strategies on the conformational equilibrium of monomeric αSyn.
Natively unstructured proteins defy the classical “one sequence–one structure” paradigm of protein science. In pathological conditions, monomers of these proteins can aggregate in the cell, a process that underlies neurodegenerative diseases such as Alzheimer and Parkinson. A key step in the aggregation process—the formation of misfolded intermediates—remains obscure. To shed light on this process, we characterized the folding and conformational diversity of αSyn, a natively unstructured protein involved in Parkinson disease, by mechanically stretching single molecules of this protein and recording their mechanical properties. These experiments permitted us to observe directly and quantify three main classes of conformations that, under in vitro physiological conditions, exist simultaneously in the αSyn sample. We found that one class of conformations, “β-like” structures, is directly related to αSyn aggregation. In fact, their relative abundance increases drastically in three different conditions known to promote the formation of αSyn fibrils. We expect that a critical concentration of αSyn with a “β-like” structure must be reached to trigger fibril formation. This critical concentration is therefore controlled by a chemical equilibrium. Novel pharmacological strategies can now be tailored to act upstream, before the aggregation process ensues, by targeting this equilibrium. To this end, single-molecule force spectroscopy can be an effective tool to tailor and test new pharmacological agents.
A single-molecule study detects structured and unstructured conformers in equilibrium in monomeric α-synuclein. The β-like conformers increase with pathological mutations and under other conditions known to promote aggregation.
Many peptides and proteins self-assemble into amyloid fibrils, including polypeptides that are associated with human amyloid diseases, mammalian and fungal prion proteins, and proteins that are believed to have biologically functional amyloid states. Proper understanding of the common propensity for polypeptides to form amyloid fibrils depends on elucidation of the molecular structures of these fibrils, as does rational design of amyloid inhibitors and imaging agents. Whereas amyloid fibril structures were largely mysterious 15 years ago, a considerable body of reliable structural information now exists, with important contributions from solid state nuclear magnetic resonance (NMR) measurements. This article reviews results from our laboratories and discusses several structural issues that have been sources of controversy.
In many cases, the molecular structures of amyloid fibrils are not determined uniquely by their amino acid sequences. Self-propagating, molecular-level polymorphism complicates the structure determination problem and can lead to apparent disagreements between results from different laboratories, when in fact different laboratories are simply studying different polymorphs. For 40-residue β-amyloid (Aβ1–40) fibrils associated with Alzheimer’s disease, we have developed detailed structural models from solid state NMR and electron microscopy data for two polymorphs, which we found to have similar peptide conformations, identical in-register parallel β-sheet organizations, but different overall symmetry. Other polymorphs have also been partially characterized by solid state NMR, and appear to have similar structures. In contrast, cryo-electron microscopy studies that use significantly different fibril growth conditions have identified structures that appear (at low resolution) to be different from those examined by solid state NMR.
The in-register parallel β-sheet organization found in β-amyloid fibrils has also been found in many other fibril-forming systems by solid state NMR and electron paramagnetic resonance (EPR), and is attributable to stabilization of amyloid structures by intermolecular interactions among like amino acids, including hydrophobic interactions and polar zippers. Surprisingly, antiparallel β-sheets have been identified and characterized by solid state NMR in certain fibrils formed by the D23N mutant of Aβ1–40, which is associated with early-onset, familial neurodegenerative disease. Antiparallel D23N-Aβ1–40 fibrils are metastable with respect to conversion to parallel structures, and therefore represent an off-pathway intermediate in the amyloid fibril formation process. Evidence for antiparallel β-sheets in other amyloid-formation intermediates has been obtained recently by other methods.
As an alternative to simple parallel and antiparallel β-sheet structures, β-helical structural models have been proposed for various fibrils, especially those formed by mammalian and fungal prion proteins. Solid state NMR and EPR data show that fibrils formed in vitro by recombinant PrP have in-register parallel β-sheet structures, but the structure of infectious PrP aggregates is not yet known definitively. The fungal HET-s prion protein has been shown by solid state NMR to have a β-helical structure, but all yeast prions studied by solid state NMR (i.e., Sup35p, Ure2p, and Rnq1p) have in-register parallel β-sheet structures, with the fibril core being formed by their Gln- and Asn-rich N-terminal segments.
To investigate the α-synuclein protein and its role in Parkinson’s disease, we screened a library of random point mutants both in vitro and in yeast to find variants in an unbiased way that could help us understand the sequence-phenotype relationship. We developed a rapid purification method that allowed us to screen 59 synuclein mutants in vitro and discovered two double point mutants that fibrillized slowly relative to wild type, A30P, and A53T α-synucleins. The yeast toxicity of all of these proteins was measured and we found no correlation with fibrillization rate, suggesting that fibrillization is not necessary for synuclein-induced yeast toxicity. We also found that β-synuclein was of intermediate toxicity to yeast and γ-synuclein was non-toxic. Coexpression of Parkinson’s disease related genes DJ-1, parkin, Pink1, UCH-L1, or synphilin, with synuclein, did not affect synuclein toxicity. A second screen, of several thousand library clones in yeast, identified 25 non-toxic α-synuclein sequence variants. Most of these contained a mutation to either proline or glutamic acid that caused a defect in membrane binding. We hypothesize that yeast toxicity is caused by synuclein binding directly to membranes at levels sufficient to non-specifically disrupt homeostasis.
synuclein; Parkinson’s disease; fibrillization; membrane binding; yeast toxicity
While most forms of Parkinson’s Disease (PD) are sporadic in nature, a small percentage of PD have genetic causes as first described for dominant, single base pair changes as well as duplication and triplication in the α-synuclein gene. The α-synuclein gene encodes a 140 amino acid residue protein that interacts with a variety of organelles including synaptic vesicles, lysosomes, endoplasmic reticulum/Golgi vesicles and, reported more recently, mitochondria. Here we examined the structural and functional interactions of human α-synuclein with brain mitochondria obtained from an early, pre-manifest mouse model for PD over-expressing human α-synuclein (ASOTg). The membrane potential in ASOTg brain mitochondria was decreased relative to wildtype (WT) mitochondria, while reactive oxygen species (ROS) were elevated in ASOTg brain mitochondria. No selective interaction of human α-synuclein with mitochondrial electron transport complexes cI-cV was detected. Monomeric human α-synuclein plus carboxyl terminally truncated forms were the predominant isoforms detected in ASOTg brain mitochondria by 2-dimensional PAGE (Native/SDS) and immunoblotting. Oligomers or fibrils were not detected with amyloid conformational antibodies. Mass spectrometry of human α-synuclein in both ASOTg brain mitochondria and homogenates from surgically resected human cortex demonstrated that the protein was full-length and postranslationally modified by N-terminal acetylation. Overall the study showed that accumulation of full-length, N-terminally acetylated human α-synuclein was sufficient to disrupt brain mitochondrial function in adult mice.
Background: The basis of the pathogenicity of the H50Q variant α-synuclein is unknown.
Results: The critical concentration of α-synuclein is decreased by 10-fold by the H50Q mutation, and its aggregation is modulated by the wild-type isoform.
Conclusion: Key effects of the H50Q mutation on the aggregation of α-synuclein can be quantified.
Significance: Our data provide insights into the mechanism of Lewy body formation in vivo.
The conversion of α-synuclein from its intrinsically disordered monomeric state into the fibrillar cross-β aggregates characteristically present in Lewy bodies is largely unknown. The investigation of α-synuclein variants causative of familial forms of Parkinson disease can provide unique insights into the conditions that promote or inhibit aggregate formation. It has been shown recently that a newly identified pathogenic mutation of α-synuclein, H50Q, aggregates faster than the wild-type. We investigate here its aggregation propensity by using a sequence-based prediction algorithm, NMR chemical shift analysis of secondary structure populations in the monomeric state, and determination of thermodynamic stability of the fibrils. Our data show that the H50Q mutation induces only a small increment in polyproline II structure around the site of the mutation and a slight increase in the overall aggregation propensity. We also find, however, that the H50Q mutation strongly stabilizes α-synuclein fibrils by 5.0 ± 1.0 kJ mol−1, thus increasing the supersaturation of monomeric α-synuclein within the cell, and strongly favors its aggregation process. We further show that wild-type α-synuclein can decelerate the aggregation kinetics of the H50Q variant in a dose-dependent manner when coaggregating with it. These last findings suggest that the precise balance of α-synuclein synthesized from the wild-type and mutant alleles may influence the natural history and heterogeneous clinical phenotype of Parkinson disease.
alpha-Synuclein (a-synuclein); Amyloid; Fibril; Parkinson Disease; Protein Aggregation; Aggregation Propensity; Fibrils Thermodynamic Stability; Polyproline II Structure
Alzheimer's, Parkinson's and Creutzfeldt-Jakob disease are associated with inappropriate protein deposition and ordered amyloid fibril assembly. Molecular chaperones, including αB-crystallin, play a role in the prevention of protein deposition.
A series of site-directed mutants of the human molecular chaperone, αB-crystallin, were constructed which focused on the flexible C-terminal extension of the protein. We investigated the structural role of this region as well as its role in the chaperone function of αB-crystallin under different types of protein aggregation, i.e. disordered amorphous aggregation and ordered amyloid fibril assembly. It was found that mutation of lysine and glutamic acid residues in the C-terminal extension of αB-crystallin resulted in proteins that had improved chaperone activity against amyloid fibril forming target proteins compared to the wild-type protein.
Together, our results highlight the important role of the C-terminal region of αB-crystallin in regulating its secondary, tertiary and quaternary structure and conferring thermostability to the protein. The capacity to genetically modify αB-crystallin for improved ability to block amyloid fibril formation provides a platform for the future use of such engineered molecules in treatment of diseases caused by amyloid fibril formation.