Article Cite This: Biochemistry 2018, 57, 5145−5158
pubs.acs.org/biochemistry
α‑Synucleins from Animal Species Show Low Fibrillation Propensities and Weak Oligomer Membrane Disruption Cagla Sahin,†,‡ Lars Kjær,† Mette Solvang Christensen,† Jannik N. Pedersen,† Gunna Christiansen,§ Adriana-Michelle Wolf Peŕ ez,† Ian Max Møller,‡ Jan J. Enghild,‡ Jan S. Pedersen,†,∥ Knud Larsen,⊥ and Daniel E. Otzen*,†,‡ †
iNANO, Aarhus University, Gustav Wieds Vej 14, DK-8000 Aarhus C, Denmark Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark § Department of Biomedicine, Aarhus University, Wilhelm Meyers Allé 4, DK-8000 Aarhus C, Denmark ∥ Department of Chemistry, Aarhus University, Langelandsgade 140, DK-8000 Aarhus C, Denmark ⊥ Department of Molecular Biology and Genetics, Aarhus University, C. F. Møllers Allé 3, DK-8000 Aarhus C, Denmark
Biochemistry 2018.57:5145-5158. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 09/07/18. For personal use only.
‡
S Supporting Information *
ABSTRACT: The intrinsically disordered protein α-synuclein (aSN) forms insoluble aggregates in the brains of Parkinson’s disease (PD) patients. Cytotoxicity is attributed to a soluble aSN oligomeric species that permeabilizes membranes significantly more than monomers and fibrils. In humans, the A53T mutation induces early onset PD and increases the level of aSN oligomerization and fibrillation propensity, but Thr53 occurs naturally in aSNs of most animals. We compared aSNs from elephant, bowhead whale, and pig with human aSN. While all three animal aSNs showed significantly weakened fibrillation, elephant aSN formed much more oligomer, and pig aSN much less, than human aSN did. However, all animal aSN oligomers showed weakened permeabilization toward anionic lipid vesicles, indicative of decreased cytotoxicity. These animal aSNs share three substitutions compared to human aSN: A53T, G68E, and V95G. We analyzed aggregation and membrane binding of all eight mutants combining these three mutations. While the G68E mutation is particularly important in weakening fibrillation and possible toxicity, the strongest effect is seen when all three mutations are present. Thus, a small number of mutations can significantly decrease aSN toxicity. α-Synuclein (aSN) is a 140-residue intrinsically disordered protein with a potential role in neurotransmitter vesicle release.1,2 Binding of aSN to anionic lipid vesicles3 or anionic micelles4 in vitro also induces α-helical structure in the Nterminal region. aSN is also associated with neuropathological conditions in both Alzheimer’s disease and Parkinson’s disease (PD):5,6 duplication and triplication of the SNCA gene lead to PD,7,8 while mutations such as A30P, E46K, H50Q, G51D, and A53T induce early onset PD.6,9−13 All these mutations except A30P are localized in α-helix 2 of aSN, formed when aSN binds to lipid vesicles.14−16 Lipid-mediated folding of aSN stabilizes and protects its target membrane.17 In turn, membrane binding prevents aSN from aggregating.18 Hence, the E46K, H50Q, G51D, and A53T mutations could also affect lipid binding, contributing to aSN pathology. Under pathological conditions, aSN adopts a β-sheet rich conformation in misfolded oligomers, fibrils, and Lewy bodies.19 Both wild-type (WT) and mutated (A30P and A53T) aSN form insoluble fibrillary aggregates with antiparallel β-sheet structure upon incubation in vitro.20,21 Aggregate formation is accelerated in both of these PD-linked © 2018 American Chemical Society
mutations;20 A53T aggregates the fastest, while A30P has a propensity to form dense bundled fibril networks that is higher than that of the WT.22 For several neurodegenerative diseases, including PD, oligomers rather than fibrils have been proposed to be the toxic species, because they can perturb and permeabilize membranes.23,24 aSN oligomers are highly stable and consist of ∼30 aSN monomers,23,25 forming a relatively compact core surrounded by a more unstructured corona.26 Hydrogen− deuterium exchange mass spectrometry (MS) identifies the protected (i.e., hydrogen-bonded) region to span residues 18− 90, while residues 1−17 and 90−140 are not significantly protected, indicating a lack of persistent structure. However, only residues 100−140 are visible in the oligomeric structure by solution nuclear magnetic resonance (NMR),27 indicating that this region is highly mobile. The N-terminal region of aSN is involved in membrane interactions in both the monoReceived: June 7, 2018 Revised: July 23, 2018 Published: August 1, 2018 5145
DOI: 10.1021/acs.biochem.8b00627 Biochemistry 2018, 57, 5145−5158
Article
Biochemistry Table 1. Biophysical Properties of aSNs from Different Animal Species species human pig whale elephant errors
mutations relative to H. sapiens − A53T, G68E, V95G A53T, G68E, V95G, N103S, Δ104, P108S, M116T A53T, G68E, V95G, L100M, N103G, D115N, M116V −
solubility
ThT levelb
% pellet after 120 h
fibrillation levels detected by EM
β-content in fibrilsc
melting temperature in DMPG (°C)d
oligomer/ total aSNe
calcein releasef
1.61 1.77 1.77
≡100 2.4 ± 1 4±2
61 ± 5 44 ± 4 47 ± 5
+++ − −
100 0 0
57 ± 1 26 ± 1 28 ± 1
3±1 1.8 ± 0.6 4 ± 1.4
≡100 − 27 ± 2
1.65
21 ± 4
31 ± 4
+
37
34 ± 1
9.7 ± 2
51 ± 3
−
∼3%
7%
−
−
∼1 °C
∼1
∼3%
a
Theoretical value obtained from the sequence, given in arbitrary units. bThT fluorescence after incubation for 60 h relative to human aSN. cFrom far-UV CD spectroscopy. Human aSN normalized to 100% and pig and whale aSN to 0%. Numbers for elephant indicate the contribution from the human aSN spectrum to the whale CD spectrum. dFrom a thermal scan of monomeric aSN in the presence of DMPG vesicles. The melting temperature is the midpoint of the first transition from the trough around 20−30 °C. eDetermined from the size of the oligomer peak relative to the sum of the oligomer and monomer peaks in a gel filtration chromatogram. Integration was performed on half-peaks (right-hand half for oligomer, left-hand half for monomer). Based on three independent experiments at four different time points in the aggregation process (0−26 h). f Calculated using eq 1 and subsequently normalized to the value of human aSN. a
meric28,29 and oligomeric states,29 and this may affect the mobility of this region of the oligomer. In contrast to the extensive studies of PD-accelerating aSN mutations, there has been little focus on mutations that delay it. We reasoned that such mutations could be identified in organisms with a life expectancy comparable to or longer than that of humans. Thr is found naturally in position 53 in all nonhuman species,30 ranging from fish to mammals, many of which are known for their longevity. In silico studies have identified 12 phylogenetic substitutions in aSN; apart from the PD-related T53A substitution, they are all in the NAC region or C-terminal tail.31 Nevertheless, they are predicted to have significant effects on the structure (backbone torsion angle) in the N-terminal region of aSN (residues 32−58), yet the connection between mutations found in humans, short-lived organisms, and long-lived organisms and the aggregative properties of aSN remains unclear. Here we examine the aggregative properties of human aSN versus aSN from three different animals, bowhead whale (Balaena mysticetus), a species known to live for up to 200 years,32 African elephant (Loxodonta africana), which has a maximum life span of 60 years under natural conditions (which compares favorably with the estimated 30-year life expectancy of paleolithic humans), and as a control for a shorter-lived organism (∼20 years), domestic pig (Sus scrofa). The three substitutions that all three animals share relative to human aSN (A53T, G68E, and V95G) completely abolish both fibrillation and oligomerization in pig and severely weaken elephant and whale oligomers’ membrane permeabilization (despite an increase in the level of oligomerization in elephant aSN). We also dissect the contributions of the three mutations and identify G68E as the main contributor. Taken together, the data presented here support the proposal that residues at position 53, 68, and 95 protect from neurodegenerative diseases compared to the human variety, via decreased aggregation propensities and weakened oligomeric vesicle permeabilization.
Extraction of Nucleic Acids and cDNA Synthesis. RNA used for molecular cloning of the SNCA gene encoding aSN was isolated from pig frontal cortex, elephant lymph node, and bowhead kidney. Tissues were pulverized in liquid nitrogen after dissection. Total RNA was isolated by the RNeasy method (Qiagen, Hilden, Germany). The integrity of the RNA samples was verified by ethidium bromide staining of the rRNA on 1% agarose gels. Synthesis of cDNA used for cloning was conducted with 5 μg of total RNA using SuperScript II RNase H− reverse transcriptase (Invitrogen, Carlsbad, CA). cDNA synthesis was initiated by heating of total RNA, oligo(dT)12−18 primer, and dNTP at 65 °C for 5 min, followed by the addition of 200 units of reverse transcriptase and then incubation at 42 °C for 50 min, followed by 70 °C for 15 min. Cloning of Pig SNCA cDNA. Pig SNCA cDNA, elephant SNCA cDNA, and bowhead SNCA cDNA were isolated using a reverse transcription polymerase chain reaction (RT-PCR) cloning approach. PCR primers for molecular cloning of bowhead SNCA were derived from genomic and transcript sequences.32 The RT-PCR mixture contained 2.5 μL of cDNA synthesized from RNA isolated from frontal cortex and pituitary gland, 1.5 mM MgCl2, 0.2 mM dNTP, SNCA sense and antisense species-specific primers (0.5 μM each) (Table S1), and 1 unit of Phusion DNA polymerase (Finnzymes, Thermo Fisher Scientific, Waltham, MA), in a total volume of 25 μL. PCR amplification was accomplished by employing the following program: denaturation at 95 °C for 2 min, 10 cycles of touchdown (−0.5 °C per cycle) at 95 °C for 20 s, 60 °C for 30 s, and 72 °C for 45 s, followed by 25 cycles of 95 °C for 20 s, 55 °C for 30 s, and 72 °C for 45 s. The PCR program was finished with an extension step at 72 °C for 5 min. Twenty-five microliters of the amplification product was applied to a 1% agarose gel and visualized after electrophoresis by ethidium bromide staining. Fluorescent bands of approximately 420 bp for elephant and bowhead SNCA and an 800 bp band for pig SNCA were isolated and eluted using the Qiaquick gel extraction kit from Qiagen. The eluted PCR products were cloned directly into the pCR TOPO 2.1 vector (Invitrogen) and sequenced in both directions. The cloned pig SNCA sequence was used as a template in a subsequent PCR mixture containing linkers for subcloning into expression vector pET11d using restriction sites BamHI and NcoI that are compatible with BclI and PciI, respectively. Primers for subcloning SNCAPCI-F and SNCA-BCL-R are listed in Table 1. Elephant cDNA and whale cDNA were cloned into
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MATERIALS AND METHODS Materials. Human aSN was purified by recombinant expression in Escherichia coli with a codon-optimized expression vector as described previously.26 All chemicals were of the highest possible purity and were from SigmaAldrich (St. Louis, MO) unless otherwise stated. Lipids were from Avanti Polar Lipids (Alabaster, AL). 5146
DOI: 10.1021/acs.biochem.8b00627 Biochemistry 2018, 57, 5145−5158
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column (GE Healthcare), and fractions containing calcein were identified by fluorescence (excitation and emission at 495 and 515 nm, respectively). Typically, the first 0.5 mL was discarded, and the second 0.5 mL was collected for the assay. Two microliters of calcein vesicles was added to 138 μL of PBS, and the fluorescence was monitored on a Varioskan Flash fluorimeter (Thermo Fisher Scientific) with excitation and emission at 495 and 515 nm, respectively, a 5 nm bandwidth, and a 100 ms measurement using top optics with autorange. Subsequently, 10 μL of the oligomer was added, and the fluorescence was measured over 40 min. Finally, 1 μL of 1% Triton X-100 was added to each well to lyse all vesicles, and the fluorescence was allowed to stabilize for 10 min. Calcein release was calculated using the equation
a pET28a vector from the previously purified SNCA by Mutagenex (Suwanee, GA) and mutated to replace any TAC Tyr codons with TAT (TAC codons can cause misincorporation of Cys into recombinant aSN33). The vectors were transformed into E. coli BL21 DE3 T1 cells by electroporation and selected with kanamycin, and aSN was expressed and purified as described previously.26 The identity was confirmed by MS analysis. The theoretical extinction coefficient of 0.412 (mg/mL)−1 known for human aSN could also be applied to all variants for concentration determination. Analysis of Fibrils. Fibrillation of 1 mg/mL aSN was performed as described previously.34 Transmission electron microscopy (TEM) pictures were recorded using phosphotungstic acid staining as described previously.35 FTIR and farultraviolet (far-UV) circular dichroism (CD) spectra were recorded as described previously26 with the modification that samples for CD were sonicated using a Qsonica sonicator with a 1/16 in. tip at 20% intensity on 5 s on/5 s off for 1 min to reduce light scattering. CD spectra were measured from 250 to 195 nm at a bandwidth of 2 nm, a scanning speed of 50 nm/ min, and a response time of 2 s using a 0.1 cm quartz cuvette. To determine the amount of soluble material left after fibrillation, samples were spun down for 10 min at 21000g and room temperature and the pellet and supernatant were run on a nonreducing 4−15% sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) gel (Bio-Rad, Hercules, CA). Gel bands were quantified using ImageJ. Binding of Monomeric aSN to Vesicles. Vesicles were made by suspending 1,2-dimyristoyl-sn-3-phosphatidylglycerol (DMPG) in phosphate-buffered saline (PBS) to a concentration of 2 mg/mL and freeze thawing the solution 10 times, followed by extrusion through a 200 nm filter 10 times.27 Solutions of 0.2 mg/mL aSN and 1 mg/mL DMPG vesicles were incubated at the start temperature for >1 min prior to measurement. CD spectra and thermal scans were recorded on a Jasco J-810 spectrometer.36 Spectral data are excluded where the PMT voltage was 10 °C does not show two transitions between 10 and 30 °C but does show only one transition coincident with the melting point of DMPG (23 °C). The trough for these mutants is much shallower and narrower than for human aSN. Relative to that of human aSN, the changes in ellipticity (pretransition level minus trough level) are only around 13, 24, and 34% for pig, whale, and elephant aSN. Also, all mutants
aSN variants, which agreed nicely with predicted monomer solubilities. At 1 mg/mL, only human aSN showed a classic sigmoidal thioflavin T (ThT) trace with fibril formation starting after ∼5 h and completed after ∼25 h (Figure 2A). Elephant aSN fibrillated to a small extent, but much less than human aSN. Even after incubation for 120 h, there were no significant increases in the ThT level for whale or pig variants of aSN. We extended these data by measuring aSN levels in the supernatant and pellet of 120 h incubated aSN samples by SDS−PAGE with densitometric scanning (Table 1). While 60% of human aSN could be pelleted as insoluble material, this figure decreased in the animal species, reaching 30% pellet for elephant aSN. Thus, although little ThT-positive material was produced by the animal aSN, a significant though reduced amount of aSN still aggregated. We confirmed the lack of significant fibril formation in the animal variants by TEM. Consistent with ThT and SDS−PAGE data, EM analysis revealed formation of wormlike aggregates for elephant aSN and amorphous aggregates for whale aSN at a concentration of 1 mg/mL, whereas the human control showed classical straight fibrils (Figure 2B). Pig aSN showed a few fibrils wrapped by amorphous species. Formation of β-sheet rich amyloid fibrils was further confirmed by CD (Figure 2C). Human aSN gives a minimum around 218 nm that is characteristic of β-sheet structures. Whale aSN and pig aSN spectra are more typical of random coil, with low absorption levels that start to increase below 210 nm (light scattering from the aggregates compromises spectral 5150
DOI: 10.1021/acs.biochem.8b00627 Biochemistry 2018, 57, 5145−5158
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Biochemistry undergo a sharp melting out transition over the 25−40 °C range, with midpoint temperatures of approximately 26, 28, and 34 °C for pig, whale, and elephant aSN, followed by a more diffuse melting out at higher temperatures. These data highlight the mutants’ reduced ability to bind to vesicles (leading to helical structure) and consequent lower thermal resistance. On the basis of these data, we can rank the vesicle binding ability of the aSN variants as follows: human ≫ elephant > whale > pig. This ranking corresponds nicely to fibrillation propensities in ThT and EM experiments and βsheet levels detected by CD. Oligomers of Elephant aSN Are Larger and More Stable and Show Decreased Levels of Membrane Permeabilization Compared to Human aSN. We next turned to an analysis of the different species’ ability to form aSN oligomers by size exclusion chromatography (SEC) (Table 1 and Figure 3A). Elephant aSN forms the largest amount of oligomer, while pig aSN forms the smallest amount. The human aSN monomer elutes at 17.5 mL, whereas the oligomer elutes at ∼12 mL. Remarkably, elephant aSN increases both the amount and the size of the oligomer species, shifting the elution volume to ∼11 mL as well as a small peak at 8−9 mL. Whale aSN forms less oligomer than human and elephant aSNs do and produces larger amounts of the degraded monomer eluting around 20 mL. A peak eluting around 20 mL contained a 103-amino acid protein, corresponding to degraded aSN according to MS studies of human aSN (C. Sahin et al., unpublished data). Porcine aSN does not form oligomers (data not shown). The SEC-purified oligomers showed no change in secondary structure as determined by CD spectroscopy (Figure S2); however, the elephant oligomers were slightly larger and more prone to associate into bundles according to TEM (Figure S3), while human aSN and whale aSN largely form spherical structures. More detailed structural information was provided by SAXS, which has previously shown human aSN oligomers to consist of ∼29 monomers forming a compact ellipsoidal core surrounded by a shell of less structured protein.23 On the basis of the forward scattering, the human aSN oligomer was estimated to have a molecular weight of 407 kDa corresponding to 28 monomers, which agreed well with our earlier estimates. While the elephant aSN oligomer also fitted the ellipsoidal model (Figure 3B and Table 2), its molecular weight was 689 ± 14 kDa, corresponding to 47 monomers (Table 2). Consistent with this, the p(r) function (Figure 3C) showed the elephant variant to have a maximum diameter (24
nm) slightly larger than that of the human aSN oligomer (20 nm) as well as larger Rg values of 8.1 ± 0.2 and 7.0 ± 0.5 nm. The fraction of protein in the flexible region of the structure is ∼0.5 for both elephant and human oligomers, showing that the core forming part of the protein has likely not changed but rather structural rearrangements in the core caused the size difference. While both the elephant and human oligomer remained intact at elevated temperatures (Figure S2), they showed different urea sensitivity. The elephant oligomer remained stable up to 8 M urea, but human aSN oligomers started to dissociate above 5 M urea (Figure S4) as observed previously.25 We examined the membrane permeabilizing effects of the oligomer variants, measured as the release of calcein from DOPG vesicles. It was not possible to purify sufficient amounts of pig aSN oligomers to perform the assays for this variant because of its low yield. Compared to human aSN oligomers at 0.1 mg/mL, elephant oligomers gave rise to only 51% release of calcein and only 27% release for the whale variant of aSN (Figure 3D). Dissection of the Properties of Individual Mutations Separating Human and Animal aSN. Having shown a consistent change in fibrillation propensity and the oligomer’s membrane perturbation across the three aSN variants, we examined the three mutations that the animals shared relative to human aSN, namely, A53T, G68E, and V95G (summarized in Table 4). All of the eight available mutant combinations (including human and pig aSN as the two end point species) were prepared, and their aggregation and vesicle permeabilization properties were determined. At 0.25 mg/mL (Figure 4A), fibrillation took place at the same rate for V95G, A53T, and A53T/V95G as for the WT, with a lag time of ≈11 h. Double mutants A53T/G68E and G68E/V95G exhibited lag times of 17 and 25 h, respectively; even slower fibrillation was observed for G68E (lag time of 50 h), and no detectable fibrillation was obtained for WT pig aSN. Similar behavior was seen at 0.5−1 mg/mL (data not shown). After fibrillation for 150 h, A53T gives rise to the most pronounced far-UV CD minimum at 218 nm, characteristic of β-sheet structure, while pig aSN retains a random coil structure (cf. Figure 2C). Deconvolution of the spectra based on linear combinations of A53T and pig A53 (Figure S5) gives the following ranking (in terms of the extent of β-sheet structure): A53T > V95G > human > G68E/V95G ≈ A53T/V95G > G68E > A53T/G68E > pig aSN. This ranking corresponds satisfactorily to the various fibrillation lag times (Figure 4A): the four fastest fibrillating mutants have the highest levels of βsheet structure along with G68E/V95E, while pig aSN and G68E have the lowest levels and the longest lag times. A53T/ G68E is a slight anomaly as it has a relatively short lag time but also rather small amounts of β-sheet structure. As depicted in Figure S6, this mutant shows the same classical unbranched fibrils as all other mutants except pig aSN (which forms no fibrils, just small amounts of amorphous aggregates) and G68E (low levels of relatively short fibrils). Cross-seeding among the eight aSN mutants was examined by seeding the WT human monomer with sonicated fibrils from the mutants (Figure 4C) and by self-seeding monomers of aSN mutants with sonicated fibrils of the same mutants (Figure 4D and Table 3). Data show a general reduction in the level of seeding compared to that of human aSN. A53T seeds WT human monomers to a significantly reduced extent. V95G seeds induce fibrillation in both the mutant and human WT
Table 2. Comparison of Parameters Obtained from IFT Analysis and from Model Fitting of SAXS Data χ2 Rg (nm) Rg chain (nm)a Nmonomer (IFT) Nmonomer (model) εb fshellc Rcore (nm) Rshell (nm)
human
elephant
0.96 7.0 ± 0.5 2.4 28 ± 1 29 ± 1 1.9 ± 0.1 0.52 3.2 ± 0.1 5.0 ± 0.2
2.3 8.1 ± 0.2 2.3 47 ± 1 51 ± 1 2.0 ± 0.1 0.49 4.0 ± 0.1 6.0 ± 0.2
a
Radius of gyration of aSN coils in solution. bWidth:height ratio of the ellipsoid. cFraction of protein in the flexible region. 5151
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Figure 4. Fibrillation and seeding of different aSN variants. (A) Fibrillation observed for the eight variants of aSN when shaken at a protein concentration of 0.25 mg/mL. Fibrillation is monitored with ThT fluorescence. (B) Degree of β-sheet structure after fibrillation for 150 h determined with CD. (C) Cross-seeding with 5% sonicated fibrils from the mutants in the WT human monomer at a concentration of 0.25 mg/mL. The fibrillation is monitored with ThT fluorescence under unshaken conditions. The error bars are standard deviations over three replicates. (D) Self-seeding with 5% sonicated fibrils from the mutants in their own monomers at a concentration of 0.25 mg/mL. The fibrillation is monitored with ThT fluorescence under unshaken conditions.
Table 3. Ability of aSN Mutants to Cross Seed Using WT aSN Monomers Together with Eight Different Fibril Seeds as Well as Self-Seeding monomer WT seed
WT A53T/V95G V95G G68E A53T A53T/G68E G68E/V95G pig aSN
+++ ++ ++ + + − − −
A53T/V95G
V95G
G68E
A53T
A53T/G68E
G68E/V95G
+ ++ − +++ ++ −
but in both cases at a reduced rate compared to that of the WT control, which seeds itself very well. G68E seeds both human WT and G68E monomers poorly. A53T/G68E does not seed well when added to the human WT monomer, because limited aggregation is obtained. However, A53T/G68E seeds itself well, though the rate of fibrillation is slower than for the human
WT. The human WT monomer seeded with A53T/V95G aggregates essentially as well as the human WT seeded with itself, though A53T/V95G seeds itself poorly. G68E/V95G does not significantly seed either the WT monomer or itself. We investigated the binding of the eight aSN variants to DMPG vesicles by far-UV CD at 37 °C (Figure 5A), which 5152
DOI: 10.1021/acs.biochem.8b00627 Biochemistry 2018, 57, 5145−5158
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Biochemistry Table 4. Different Properties for the aSN Variantsa
ThT lag time (h)b ThT end signalc (1 mg/mL) β-sheet content CD spectrum as a fraction of A53Td cross-seeding with the WT monomer (lag phase) self-seeding ability lipid binding oligomer formation vesicle permeabilization
WT (human)
V95G
A53T/ V95G
A53T/ G68E
G68E/ V95G
G68E
A53T
A53T/G68E/V95G (pig)
11 +++ +++ 0.71 +++ +++ +++ ++ ++
11 +++ +++ 0.84 ++ ++ +++ ++ ++
11 +++ ++ 0.55 ++ + ++ +++ +++
17 +++ + 0.16 − ++ +++ +++ +
25 ++ ++ 0.62 − − +++ +++ +
50 + + 0.23 + − +++ ++ +
11 +++ +++ 1.00 + +++ ++ ++ +
− − − 0.00 − − − − N/A
Legend: +++, the strongest signal; ++, a medium signal; +, a weaker signal; −, no signal. bThe time it takes for aSN to show a significant increase in ThT fluorescence intensity compared to the baseline level. cThe level of ThT intensity in its saturated phase (end point). dβ-Sheet content normalized to the β-sheet content of A53T-based CD spectra. a
Figure 5. Lipid interactions of aSN variants as monomers and oligomers. (A) Structual analysis of aSN structure formation by wavelength scans on DMPG vesicle surfaces at 37 °C using CD. (B) CD analysis of the development of structure over temperature on DMPG vesicles. The inset shows the correlation between the depth of the trough and the midpoint temperature of melting for the 40−70 °C temperature range. (C) Oligomer formation in aSN mutants upon 5 h incubations of 3 mg/mL solutions over two separate experimental analyses. (D) Calcein release by oligomers of the various mutants at 0.2 μM (monomer units).
°C temperature range (inset of Figure 5B), indicating that the stronger the aSN binds to the lipid, the more it is stabilized against denaturation.47 The mutants generally follow the trend provided by the individual animal aSN investigated in Figure 2D. Oligomers were purified from each of the eight mutants, and the amount of oligomer/monomer was quantified (Figure 5C). The formation of oligomers varies between experimental runs,
showed a slight reduction in the amount of induced structure in the following order: WT > G68E/V95G > G68E > V95G > A53T/G68E > A53T/V95G > A53T ≫ pig. Pig aSN undergoes no significant change in structure in the presence of DMPG vesicles, suggesting a very low level of binding. The ranking is confirmed by temperature scans (Figure 5B), which also show a significant correlation between the depth of the trough and the midpoint temperature of melting in the 40−70 5153
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Congo red stain assays as well as by TEM. Minor sequence variations were identified in the C-terminal region that makes up the fibril core of the prion proteins, but these do not have a major impact on prion aggregation properties. This contrasts to the results of our studies in which sequence alignment identified one residue at position 68 as being crucial for the fibrillation and oligomerization properties of aSN. Residue 68 in aSN is located in the NAC region, which is involved both in membrane interactions and in the formation of the hydrophobic fibril core. A Correlation among α-Helical Structure, Membrane Binding, and Aggregation Propensity. What consequence does this have in vivo? While aSN’s natural function is not yet firmly established, it is probably related to the mobilization and formation of signaling reserve vesicles,49 consistent with the formation of α-helical structures on the surface of lipid vesicles. Interestingly, for the different animal species, the extent of fibrillation correlates with the level of α-helix formation, with the most fibrillation prone version of aSN (human, followed by elephant) also showing the most α-helical structure and, in turn, being the most stabilized against thermal unfolding. Whale and pig aSNs show very little lipid binding propensity, despite the lack of substitutions in the 50 N-terminal residues involved in lipid contacts. Decreasing the percentage of anionic lipid to mimic the composition of biological membranes decreases both the helicity and the melting transition temperature even further.47 Thus, helicity can be considered a measure of the degree of binding of aSN to anionic phospholipid vesicles, which in turn could suggest a positive link between lipid binding and fibrillation. This is also confirmed by the human A30P mutant that binds more poorly to lipids than WT human aSN50,51 and fibrillates more slowly;52 its ability to induce early onset PD is attributed to its enhanced oligomerization propensity.53 Note that the non-human aSN structures all lose their helical structure in the presence of DMPG vesicles well below the body temperature of the animals involved, i.e., 35.6 °C for elephant,54 33.8 °C for bowhead whale,55 and 37.1 °C for pig.56 This suggests that most of the helical structure will not be present under physiological conditions even under optimal lipid binding conditions. Remarkably, the degree of oligomerization varied across the different aSN variants, with elephant aSN forming significantly more oligomer than WT human aSN and pig aSN forming significantly less, yet these animal oligomers show strongly reduced levels of calcein release, indicating low cell toxicity consistent with the low level of monomeric aSN binding to vesicles. This makes the large (>10%) fraction of the elephant aSN oligomer intriguing. It is not likely to be toxic to membranes, though it conforms to our previously reported overall core−shell oligomer model for human aSN with slightly larger dimensions. The reduced level of membrane binding is likely linked to the monomer’s poor lipid binding, assuming that the same regions of aSN are involved in lipid contacts in the monomer and oligomer. Nontoxic oligomers could constitute a sink for aSN that is just as innocuous as fibrils but could be mobilized more readily for the release of monomeric aSN. Importance of A53, G68, and V95 in Fibrillation, Oligomerization, and Toxicity. The point mutations in positions 53, 68, and 95 shared by all three animals are predicted to increase the solubility of monomeric aSN compared to that of human aSN and thus decrease the relative stability of the insoluble state. The stability of the fibrillary
but there are some consistent trends. (1) Human WT, A53T, G68E, and V95G broadly form the same levels of oligomers. (2) G68E/V95G shows a slightly higher level of oligomer formation. (3) A53T/G68E and A53T/V95G show 2−6-fold higher levels of oligomer formation. The membrane permeabilization ability of the oligomers also varies among the different mutants, with A53T, G68E, A53T/ G68E, and G68E/V95G showing approximately half the permeabilization ability of human aSN in the calcein release assays, V95G showing the same permeabilization as the human WT, and A53T/V95G showing approximately twice as much permeabilization (Figure 5D). When both the oligomer concentration and membrane permeabilization are taken into consideration, A53T/G68E gives rise to more aggregate toxicity as the increased amount of oligomer formed outweighs the reduced level of permeabilization. The weakened membrane interaction could be explained by a change in secondary structure that affects the oligomer’s membrane binding properties. A53T/V95G is formed in larger amounts and leads to higher levels of permeabilization, suggesting that it would be much more toxic than the human WT.
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DISCUSSION Weakened Fibrillation in Animal Species Centers around Position 68. Because several animal species lived significantly longer than humans prior to the emergence of modern civilization, they may be expected to have evolved to withstand old age better than humans; e.g., in protein the level of fibrillation diseases where the sequence of the offending protein is a straightforward target for evolution. We do not claim that a lack of fibrillation by itself leads to extended life spans but suggest that animals that in various ways have evolved to have long life spans have also had to deal with the challenge of protein aggregation. (The remarkably low fibrillation and oligomerization properties of porcine aSN, derived from a relatively short-lived species, illustrate that short-lived species do not necessarily possess highly fibrillogenic aSN; in contrast, highly fibrillogenic aSN would a priori not be compatible with a long life.) Humans are not on a particularly long-lived branch of the mammalian family tree. Their closest relatives are apes (chimpanzee, maximum age in the wild, 50−60 years; orangutan, 59 years), monkeys (Bolivian spider monkey, 18 years; white cheek gibbon, 28 years; American spider monkey, 47 years), shrews (Chinese tree shrew, 3 years; tree shrew, 10 years), bats (6−30 years) lemurs (27 years), and mice (4 years). Most longer-lived animals on this branch share residues A53 and V95 with humans.30 The A53T mutation in human aSN leads to an increased oligomerization toxicity and familial early onset PD but occurs in all other examined species ranging from fish to mammals, several of which are known for their longevity, e.g., the bowhead whale (200 years) and large land mammals (camels, 50 years; elephants, 60 years). The protective mutations present in our specific branch of the tree of life center around position 68, which is Gly on the human branch but Glu in most animals. Indeed, our fibrillation experiments, confirmed by CD, SDS−PAGE, and EM, showed substantially weakened fibrillation by all of four animal aSNs. In a similar manner, Nyström and Hammarström compared prion proteins from several mammalian species, including humans, in relation to prion-resistant species such as pig.48 However, prions from all species seemed to fibrillate as monitored by ThT and 5154
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Membrane Binding Is Driven by Multiple Regions of aSN. It is unclear what advantage the much more fibrillation prone human aSN may have compared to pig aSN, though this may relate to the elusive function of aSN in vivo. The ability to form α-helical structures on DMPG membranes declines for all the mutations, particularly driven by the A53T mutation that itself reduces the degree of structural change by at least 2-fold. Because whale and elephant, which are both long-lived and have all these three mutations as well as several others, show lipid binding that is better than that of pig aSN, it may be that these extra mutations (all in the C-terminal part of the protein) compensate for the poor membrane binding of the central helix. Both elephant and whale have mutations in the Cterminal part that reduce the degree of negative charge (D115N or deletion of E104) and would weaken the repulsion of anionic vesicles. Native membrane binding features are thus weaker in pig aSN, though further insight requires additional analysis of membrane binding by several other naturally occurring mutants. In principle, levels of aSN expression patterns and thus different levels of partitioning to lipids could also play a role. However, we note that the expression of aSN in the lizard Anolis carolinensis is very similar to that in both humans and mice,58 indicating that the expression levels do not vary greatly across species. Inhibition of WT Human aSN Fibrillation by CrossSeeding with Pig aSN. Given the potential for gene therapy, and possibly fibrillation-resistant neuron replacement therapy, we examined the extent to which it is possible to cross-seed the human WT with the aSN mutants. It is not possible to seed monomeric pig aSN with fibril seeds made of any of the mutants, which emphasizes the severe reduction in pig aSN fibrillation propensity. We also found that relatively few of the mutants can be used to cross-seed WT human aSN. A53T/ V95G and V95G cross-seed most effectively with the human WT, whereas none of the mutants containing the G68E mutation show any significant cross-seeding. This suggests that even when there is the capacity to fibrillate (A53T/G68E fibrillates quite readily), this mutation prevents the extension of the fibrils by other aSN species that do not have the same amino acid sequence. Thus, relatively minor changes render seeding ineffective, consistent with the observation that mouse aSN functions as a competitive inhibitor for the fibrillation of human aSN.58 Also, the mutants undergo limited self-seeding only under non-agitated conditions, with A53T/V95G monomers seeding WT fibrils much better than A53T/V95G fibrils. Thus, WT fibrils may represent a more stable fibrillary structure that can imprint other monomers that would otherwise not form such fibrils. In summary, aSNs from whale, elephant, and pig show extremely low levels of fibrillation and low oligomer toxicity levels. This is a strong indication that aSN’s propensity to form fibrils and toxic oligomers is a problem specific to the branch of the tree of life on which humans reside. Our mutation study indicates that this might be because of the loss of a trio of conserved amino acids (A53T, G68E, and V95G) that have cooperative protective effects. All these residues are found in the most structured part of the aSN oligomer59 and fibrils,57 suggesting that a comparative analysis of the structure and dynamics of oligomers and fibrils from different species may provide further insight into the molecular basis for aggregate toxicity.
species can also be affected more directly. The solid-state NMR structure of human aSN fibrils shows Gly68 and Val95 to be in close contact (bridging the two ends of the Greek key motif, though the Cα atomm of Gly68 is pointing away from Val95), with Ala53 in a completely separate part of the fibril.57 Modifications at positions 68−95 likely perturb fibril structure, in particular introduction of a polar moiety in position 68 that is placed in a position where the fibrils undergo a sharp 90° turn and thus require the flexibility provided by Gly at this position; in contrast, the Gly95 substitution introduces a strand-destabilizing side chain into the middle of an existing βsheet. Rather than a regular decrease in the level of fibrillation and oligomer toxicity from the highly fibrillogenic human aSN to the nonfibrillogenic pig aSN, we find that all mutations that increase the lag time of aSN fibrillation include G68E. Halflives increase in the following order: A53T/G68E < G68E/ V95G ≪ G68E ≪ A53T/G68E/V95G (pig). None of the mutants (e.g., A53T and V95G) fibrillate faster or significantly more extensively than WT human aSN. This suggests that the G68E mutation is vital for the other mutations to have a protective effect against fibrillation and may thus counteract the effect of A53T. This is partially achieved for the G68E/ A53T mutation and even more for the G68E/V95G mutation, but all three mutations (leading to pig aSN) are needed to fully prevent fibrillation and oligomerization. A53T/V95G (in a Gly68 background) shows by far the strongest oligomer membrane permeabilization (weighted as a combination of oligomer propensity and membrane permeabilization). A single mutation that introduces Gly at position 68 might dramatically increase both the oligomer’s membrane perturbation properties and the propensity to fibrillate. As position 68 is Glu in the whole branch where the human variant resides (except for Gly68 in humans), the remaining mutations found in humans could be a positive selective trait to counteract the effects of Gly68. Apes (including humans) have lost the 53T in a parallel event with the Bolivian squirrel monkey. Also, 95G has been either turned into a Val in multiple different events or reintroduced in some lines. In addition, while we see multiple events leading to reduced oligomer toxicity as we approach the human sequence on the upper branch, there is not a single instance of mutations at amino acids 53 and 95 in the species on the lower branch where 68E is intact, and the positive selection pressure is therefore maintained. This suggests that the protective features of the three mutations are evolutionarily limited by requiring all of them to provide any significant improvement. G68E is not particularly toxic on its own, and it alleviates the ability of the remaining mutations to permeabilize membranes. Substitution of Glu68 would quickly lead to either (A) a reverse mutation or (B) the loss of either of the two remaining mutations in a long-lived species. As the life span of creatures fluctuates significantly over evolutionary history, one could imagine such a mutation happening for a creature with a short life span, where a descendant would have a longer life span. Once this happens, the negative effects of substituting Glu at position 68 make the loss of the other pig amino acids favorable, and as this happens, it becomes increasingly unlikely that this function will be reestablished at a later date as longerliving descendants emerge. Evolutionarily, this creates a “valley of death” where several useless or directly harmful mutations have to take place in the longer-living species on the one branch of the tree of life to evolve toward the more advantageous version on the other. 5155
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Cookson, M. R., Muenter, M., Baptista, M., Miller, D., Blancato, J., Hardy, J., and Gwinn-Hardy, K. (2003) alpha-Synuclein locus triplication causes Parkinson’s disease. Science 302, 841. (8) Chartier-Harlin, M. C., Kachergus, J., Roumier, C., Mouroux, V., Douay, X., Lincoln, S., Levecque, C., Larvor, L., Andrieux, J., Hulihan, M., Waucquier, N., Defebvre, L., Amouyel, P., Farrer, M., and Destee, A. (2004) Alpha-synuclein locus duplication as a cause of familial Parkinson’s disease. Lancet 364, 1167−1169. (9) Kruger, R., Kuhn, W., Muller, T., Woitalla, D., Graeber, M., Kosel, S., Przuntek, H., Epplen, J. T., Schols, L., and Riess, O. (1998) Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson’s disease. Nat. Genet. 18, 106−108. (10) Appel-Cresswell, S., Vilarino-Guell, C., Encarnacion, M., Sherman, H., Yu, I., Shah, B., Weir, D., Thompson, C., Szu-Tu, C., Trinh, J., Aasly, J. O., Rajput, A., Rajput, A. H., Jon Stoessl, A., and Farrer, M. J. (2013) Alpha-synuclein p.H50Q, a novel pathogenic mutation for Parkinson’s disease. Mov. Disord. 28, 811−813. (11) Zarranz, J. J., Alegre, J., Gomez-Esteban, J. C., Lezcano, E., Ros, R., Ampuero, I., Vidal, L., Hoenicka, J., Rodriguez, O., Atares, B., Llorens, V., Gomez Tortosa, E., del Ser, T., Munoz, D. G., and de Yebenes, J. G. (2004) The new mutation, E46K, of alpha-synuclein causes Parkinson and Lewy body dementia. Ann. Neurol. 55, 164− 173. (12) Fares, M. B., Ait-Bouziad, N., Dikiy, I., Mbefo, M. K., Jovicic, A., Kiely, A., Holton, J. L., Lee, S. J., Gitler, A. D., Eliezer, D., and Lashuel, H. A. (2014) The novel Parkinson’s disease linked mutation G51D attenuates in vitro aggregation and membrane binding of alpha-synuclein, and enhances its secretion and nuclear localization in cells. Hum. Mol. Genet. 23, 4491−4509. (13) Lesage, S., Anheim, M., Letournel, F., Bousset, L., Honore, A., Rozas, N., Pieri, L., Madiona, K., Durr, A., Melki, R., Verny, C., and Brice, A. (2013) G51D alpha-synuclein mutation causes a novel parkinsonian-pyramidal syndrome. Ann. Neurol. 73, 459−471. (14) Bussell, R., Ramlall, T. F., and Eliezer, D. (2005) Helix periodicity, topology, and dynamics of membrane-associated alphasynuclein. Protein Sci. 14, 862−872. (15) Fusco, G., De Simone, A., Gopinath, T., Vostrikov, V., Vendruscolo, M., Dobson, C. M., and Veglia, G. (2014) Direct observation of the three regions in alpha-synuclein that determine its membrane-bound behaviour. Nat. Commun. 5, 3827. (16) Chandra, S., Chen, X., Rizo, J., Jahn, R., and Südhof, T. C. (2003) A broken α-helix in folded α-synuclein. J. Biol. Chem. 278, 15313−15318. (17) Lokappa, S. B., Suk, J. E., Balasubramanian, A., Samanta, S., Situ, A. J., and Ulmer, T. S. (2014) Sequence and membrane determinants of the random coil-helix transition of alpha-synuclein. J. Mol. Biol. 426, 2130−2144. (18) Zhu, M., and Fink, A. L. (2003) Lipid binding inhibits alphasynuclein fibril formation. J. Biol. Chem. 278, 16873−16877. (19) Uversky, V. N. (2007) Neuropathology, biochemistry, and biophysics of alpha-synuclein aggregation. J. Neurochem. 103, 17−37. (20) Narhi, L., Wood, S. J., Steavenson, S., Jiang, Y., Wu, G. M., Anafi, D., Kaufman, S. A., Martin, F., Sitney, K., Denis, P., Louis, J. C., Wypych, J., Biere, A. L., and Citron, M. (1999) Both familial Parkinson’s disease mutations accelerate alpha-synuclein aggregation. J. Biol. Chem. 274, 9843−9846. (21) Conway, K. A., Harper, J. D., and Lansbury, P. T. (2000) Fibrils formed in vitro from alpha-synuclein and two mutant forms linked to Parkinson’s diseases are typical amyloid. Biochemistry 39, 2552−2563. (22) Nielsen, S. B., Macchi, F., Raccosta, S., Langkilde, A. E., Giehm, L., Kyrsting, A., Svane, A. S., Manno, M., Christiansen, G., Nielsen, N. C., Oddershede, L., Vestergaard, B., and Otzen, D. E. (2013) Wildtype and A30P mutant alpha-synuclein form different fibril structures. PLoS One 8, e67713. (23) Lorenzen, N., Nielsen, S. B., Buell, A. K., Kaspersen, J. D., Arosio, P., Vad, B. S., Paslawski, W., Christiansen, G., ValnickovaHansen, Z., Andreasen, M., Enghild, J. J., Pedersen, J. S., Dobson, C. M., Knowles, T. P., and Otzen, D. E. (2014) The role of stable alpha-
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.8b00627. Oligonucleotide primers used in cloning of the SNCA cDNAs (Table S1), intrinsic solubility scores (Figure S1), far-UV CD spectra of oligomers from 20 to 90 °C (Figure S2), additional oligomer data in the form of TEM images (Figure S3) and urea stability (Figure S4), deconvolution of fibril CD spectra (Figure S5), and TEM images of fibril end point samples (Figure S6) (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Daniel E. Otzen: 0000-0002-2918-8989 Author Contributions
C.S. and L.K. contributed equally to this work. L.K. and D.E.O. conceived and designed experiments. C.S., L.K., M.S.C., J.N.P., and K.L. performed experiments. G.C., A.-M.W.P., I.M.M., J.J.E., and J.S.P. analyzed data. L.K., C.S., and D.E.O. wrote the manuscript. Funding
D.E.O. and L.K. are supported by the Danish Research Council | Natural Sciences (Grant 12-127028) and the Parkinson Society of Denmark. C.S. and I.M.M. are supported by the Danish Research Council | Technology and Production (Grant 19725). Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Chandra, S., Gallardo, G., Fernandez-Chacon, R., Schluter, O. M., and Sudhof, T. C. (2005) Alpha-synuclein cooperates with CSPalpha in preventing neurodegeneration. Cell 123, 383−396. (2) Bendor, J. T., Logan, T. P., and Edwards, R. H. (2013) The function of alpha-synuclein. Neuron 79, 1044−1066. (3) Georgieva, E. R., Ramlall, T. F., Borbat, P. P., Freed, J. H., and Eliezer, D. (2008) Membrane-bound alpha-synuclein forms an extended helix: Long-distance pulsed ESR measurements using vesicles, bicelles, and rodlike micelles. J. Am. Chem. Soc. 130, 12856−12857. (4) Rao, J. N., Jao, C. C., Hegde, B. G., Langen, R., and Ulmer, T. S. (2010) A combinatorial NMR and EPR approach for evaluating the structural ensemble of partially folded proteins. J. Am. Chem. Soc. 132, 8657−8668. (5) Uéda, K., Fukushima, H., Masliah, E., Xia, Y., Iwai, A., Yoshimoto, M., Otero, D. A., Kondo, J., Ihara, Y., and Saitoh, T. (1993) Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease. Proc. Natl. Acad. Sci. U. S. A. 90, 11282−11286. (6) Polymeropoulos, M. H., Lavedan, C., Leroy, E., Ide, S. E., Dehejia, A., Dutra, A., Pike, B., Root, H., Rubenstein, J., Boyer, R., Stenroos, E. S., Cahdrasekarappa, S., Athanassiadou, A., Papapetropoulos, T., Johnson, W. G., Lazzarini, A. M., Duvoisin, R. C., Di Iorio, G., Golbe, L. I., and Nussbaum, R. L. (1997) Mutation in the alpha-synuclein gene identified in families with Parkinson’s Disease. Science 276, 2045−2047. (7) Singleton, A. B., Farrer, M., Johnson, J., Singleton, A., Hague, S., Kachergus, J., Hulihan, M., Peuralinna, T., Dutra, A., Nussbaum, R., Lincoln, S., Crawley, A., Hanson, M., Maraganore, D., Adler, C., 5156
DOI: 10.1021/acs.biochem.8b00627 Biochemistry 2018, 57, 5145−5158
Article
Biochemistry
ray Scattering and High Resolution X-ray Diffraction. J. Appl. Crystallogr. 41, 1134−1139. (39) Glatter, O. (1977) A new method for the evaluation of smallangle scattering data. J. Appl. Crystallogr. 10, 415−421. (40) Pedersen, J. S., Hansen, S., and Bauer, R. (1994) The aggregation behavior of zinc-free insulin studied by small-angle neutron scattering. Eur. Biophys. J. 22, 379−389. (41) Oliveira, C. L., Behrens, M. A., Pedersen, J. S., Erlacher, K., Otzen, D., and Pedersen, J. S. (2009) A SAXS study of glucagon fibrillation. J. Mol. Biol. 387, 147−161. (42) Pedersen, J. S., and Svaneborg, C. (2002) Scattering from block copolymer micelles. Curr. Opin. Colloid Interface Sci. 7, 158−166. (43) Kohn, J. E., Millett, I. S., Jacob, J., Zagrovic, B., Dillon, T. M., Cingel, N., Dothager, R. S., Seifert, S., Thiyagarajan, P., Sosnick, T. R., Hasan, M. Z., Pande, V. S., Ruczinski, I., Doniach, S., and Plaxco, K. W. (2004) Random-coil behavior and the dimensions of chemically unfolded proteins. Proc. Natl. Acad. Sci. U. S. A. 101, 12491−12496. (44) Sormanni, P., Aprile, F. A., and Vendruscolo, M. (2015) The CamSol method of rational design of protein mutants with enhanced solubility. J. Mol. Biol. 427, 478−490. (45) Gasteiger, E., Gattiker, A., Hoogland, C., Ivanyi, I., Appel, R. D., and Bairoch, A. (2003) ExPASy: The proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res. 31, 3784−3788. (46) Du, H. N., Tang, L., Luo, X. Y., Li, H. T., Hu, J., Zhou, J. W., and Hu, H. Y. (2003) A peptide motif consisting of glycine, alanine, and valine is required for the fibrillization and cytotoxicity of human alpha-synuclein. Biochemistry 42, 8870−8878. (47) Kjær, L., Giehm, L., Heimburg, T., and Otzen, D. E. (2009) The influence of vesicle composition and size on α-synuclein structure and stability. Biophys. J. 96, 2857−2870. (48) Nystrom, S., and Hammarstrom, P. (2015) Generic amyloidogenicity of mammalian prion proteins from species susceptible and resistant to prions. Sci. Rep. 5, 10101. (49) Williams, R. S., and Bate, C. (2016) An in vitro model for synaptic loss in neurodegenerative diseases suggests a neuroprotective role for valproic acid via inhibition of cPLA2 dependent signalling. Neuropharmacology 101, 566−575. (50) Pantusa, M., Vad, B., Lillelund, O., Kjaer, L., Otzen, D., and Bartucci, R. (2016) Alpha-synuclein and familial variants affect the chain order and the thermotropic phase behavior of anionic lipid vesicles. Biochim. Biophys. Acta, Proteins Proteomics 1864, 1206−1214. (51) Jensen, P. H., Nielsen, M. S., Jakes, R., Dotti, C. G., and Goedert, M. (1998) Binding of alpha-synuclein to brain vesicles is abolished by familial Parkinson’s disease mutation. J. Biol. Chem. 273, 26292−26294. (52) Conway, D., Lee, S. J., Rochet, J.-C., Ding, T. T., Williamson, R. E., and Lansbury, P. T. (2000) Acceleration of oligomerization, not fibrillization, is a shared property of both alpha-synuclein mutations linked to early-onset Parkinson’s disease: implications for pathogenesis and therapy. Proc. Natl. Acad. Sci. U. S. A. 97, 571−576. (53) Li, J., Uversky, V. N., and Fink, A. L. (2001) Effect of familial Parkinson’s disease point mutations A30P and A53T on the structural properties, aggregation, and fibrillation of human alpha-synuclein. Biochemistry 40, 11604−11613. (54) Benedict, F. G., and Lee, R. C. (1936) Studies on the Body Temperature of Elephants. Proc. Natl. Acad. Sci. U. S. A. 22, 405−408. (55) Keil, G., Cummings, E., and de Magalhães, J. P. (2015) Being cool: how body temperature influences ageing and longevity. Biogerontology 16, 383−397. (56) Harig, F., Schmidt, J., Hoyer, E., Eckl, S., Adamek, E., Ertel, D., Nooh, E., Amann, K., Weyand, M., and Ensminger, S. M. (2011) Long-term evaluation of a selective retrograde coronary venous perfusion model in pigs (Sus scrofa domestica). Comp. Med. 61, 150− 157. (57) Tuttle, M. D., Comellas, G., Nieuwkoop, A. J., Covell, D. J., Berthold, D. A., Kloepper, K. D., Courtney, J. M., Kim, J. K., Barclay, A. M., Kendall, A., Wan, W., Stubbs, G., Schwieters, C. D., Lee, V. M., George, J. M., and Rienstra, C. M. (2016) Solid-state NMR structure
synuclein oligomers in the molecular events underlying amyloid formation. J. Am. Chem. Soc. 136, 3859−3868. (24) Winner, B., Jappelli, R., Maji, S. K., Desplats, P. A., Boyer, L., Aigner, S., Hetzer, C., Loher, T., Vilar, M., Campioni, S., Tzitzilonis, C., Soragni, A., Jessberger, S., Mira, H., Consiglio, A., Pham, E., Masliah, E., Gage, F. H., and Riek, R. (2011) In vivo demonstration that alpha-synuclein oligomers are toxic. Proc. Natl. Acad. Sci. U. S. A. 108, 4194−4199. (25) Paslawski, W., Andreasen, M., Nielsen, S. B., Lorenzen, N., Thomsen, K., Kaspersen, J. D., Pedersen, J. S., and Otzen, D. E. (2014) High stability and cooperative unfolding of alpha-synuclein oligomers. Biochemistry 53, 6252−6263. (26) Lorenzen, N., Nielsen, S. B., Buell, A. K., Kaspersen, J. D., Arosio, P., Vad, B. S., Paslawski, W., Christiansen, G., ValnickovaHansen, Z., Andreasen, M., Enghild, J. J., Pedersen, J. S., Dobson, C. M., Knowles, T. J., and Otzen, D. E. (2014) The role of stable αsynuclein oligomers in the molecular events underlying amyloid formation. J. Am. Chem. Soc. 136, 3859−3868. (27) Lorenzen, N., Nielsen, S. B., Yoshimura, Y., Andersen, C. B., Betzer, C., Vad, B. S., Kaspersen, J. D., Christiansen, G., Pedersen, J. S., Jensen, P. H., Mulder, F. A., and Otzen, D. E. (2014) How epigallogatechin gallate can inhibit α-synuclein oligomer toxicity in vitro. J. Biol. Chem. 289, 21299−21310. (28) Fusco, G., Chen, S. W., Williamson, P. T. F., Cascella, R., Perni, M., Jarvis, J. A., Cecchi, C., Vendruscolo, M., Chiti, F., Cremades, N., Ying, L., Dobson, C. M., and De Simone, A. (2017) Structural basis of membrane disruption and cellular toxicity by alpha-synuclein oligomers. Science 358, 1440−1443. (29) Lorenzen, N., Lemminger, L., Pedersen, J. N., Nielsen, S. B., and Otzen, D. E. (2014) The N-terminus of alpha-synuclein is essential for both monomeric and oligomeric interactions with membranes. FEBS Lett. 588, 497−502. (30) Larsen, K., Hedegaard, C., Bertelsen, M. F., and Bendixen, C. (2009) Threonine 53 in alpha-synuclein is conserved in long-living non-primate animals. Biochem. Biophys. Res. Commun. 387, 602−605. (31) Siddiqui, I. J., Pervaiz, N., and Abbasi, A. A. (2016) The Parkinson Disease gene SNCA: Evolutionary and structural insights with pathological implication. Sci. Rep. 6, 24475. (32) Keane, M., Semeiks, J., Webb, A. E., Li, Y. I., Quesada, V., Craig, T., Madsen, L. B., van Dam, S., Brawand, D., Marques, P. I., Michalak, P., Kang, L., Bhak, J., Yim, H. S., Grishin, N. V., Nielsen, N. H., Heide-Jorgensen, M. P., Oziolor, E. M., Matson, C. W., Church, G. M., Stuart, G. W., Patton, J. C., George, J. C., Suydam, R., Larsen, K., Lopez-Otin, C., O’Connell, M. J., Bickham, J. W., Thomsen, B., and de Magalhães, J. P. (2015) Insights into the evolution of longevity from the bowhead whale genome. Cell Rep. 10, 112−122. (33) Masuda, M., Dohmae, N., Nonaka, T., Oikawa, T., Hisanaga, S., Goedert, M., and Hasegawa, M. (2006) Cysteine misincorporation in bacterially expressed human alpha-synuclein. FEBS Lett. 580, 1775− 1779. (34) Giehm, L., and Otzen, D. E. (2010) Strategies to increase the reproducibility of α-synuclein fibrillation in plate reader assays. Anal. Biochem. 400, 270−281. (35) Mohammad-Beigi, H., Morshedi, D., Shojaosadati, S. A., Pedersen, J. N., Marvian, A. T., Aliakbari, F., Christiansen, G., Pedersen, J. S., and Otzen, D. E. (2016) Gallic Acid Loaded onto Polyethylenimine-Coated Human Serum Albumin Nanoparticles (PEI-HSA-GA NPs) Stabilizes α-Synuclein in the Unfolded Conformation and Inhibits Aggregation. RSC Adv. 6, 85312−85323. (36) Pantusa, M., Vad, B., Lillelund, O., Kjær, L., Otzen, D. E., and Bartucci, R. (2016) Alpha-synuclein and familial variants affect the chain order and the thermotropic phase behavior of anionic lipid vesicles. Biochim. Biophys. Acta, Proteins Proteomics 1864, 1206−1214. (37) Pedersen, J. (2004) A flux- and background-optimized version of the NanoSTAR small-angle X-ray scattering camera for solution scattering. J. Appl. Crystallogr. 37, 369−380. (38) Li, Y., Beck, R., Huang, T., Choi, M. C., and Divinagracia, M. (2008) Scatterless Hybrid Metal-Single Crystal Slit for Small Angle X5157
DOI: 10.1021/acs.biochem.8b00627 Biochemistry 2018, 57, 5145−5158
Article
Biochemistry of a pathogenic fibril of full-length human alpha-synuclein. Nat. Struct. Mol. Biol. 23, 409−415. (58) Toni, M., Cioni, C., De Angelis, F., and di Patti, M. C. (2016) Synuclein expression in the lizard Anolis carolinensis. J. Comp. Physiol., A 202, 577−595. (59) Paslawski, W., Mysling, S., Thomsen, K., Jørgensen, T. J. D., and Otzen, D. E. (2014) Co-existence of two different α-synuclein oligomers with different core structures determined by Hydrogen/ Deuterium Exchange Mass Spectrometry. Angew. Chem., Int. Ed. 53, 7560−7563.
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DOI: 10.1021/acs.biochem.8b00627 Biochemistry 2018, 57, 5145−5158
