Temperature-dependent structural changes of Parkinson's alpha-synuclein reveal the role of pre-existing oligomers in alpha-synuclein fibrillization

Abstract

Amyloid fibrils of α-synuclein are the main constituent of Lewy bodies deposited in substantial nigra of Parkinson's disease brains. α-Synuclein is an intrinsically disordered protein lacking compact secondary and tertiary structures. To enhance the understanding of its structure and function relationship, we utilized temperature treatment to study α-synuclein conformational changes and the subsequent effects. We found that after 1 hr of high temperature pretreatment, >80°C, α-synuclein fibrillization was significantly inhibited. However, the temperature melting coupled with circular dichroism spectra showed that α-synuclein was fully reversible and the NMR studies showed no observable structural changes of α-synuclein after 95°C treatment. By using cross-linking and analytical ultracentrifugation, rare amount of pre-existing α-synuclein oligomers were found to decrease after the high temperature treatment. In addition, a small portion of C-terminal truncation of α-synuclein also occurred. The reduction of pre-existing oligomers of α-synuclein may contribute to less seeding effect that retards the kinetics of amyloid fibrillization. Overall, our results showed that the pre-existing oligomeric species is a key factor contributing to α-synuclein fibrillization. Our results facilitate the understanding of α-synuclein fibrillization.

Figure 1. High temperature pretreatment inhibits α-synuclein fibril formation.

(A) α-Synuclein (25 µM) was subjected to 1 hr pretreatment in 20 (•), 40 (▴), 60 (▪), 80 (⋄), and 95 (▹) °C and the fibril formation was monitored at room temperature by addition of 20 µM ThT with continuous shaking at 1,000 rpm for 10 days. (B) Western blotting of the end-point products of α-synuclein in ThT assay. The pretreated temperatures and the molecular weight markers are indicated.

Figure 2. Fewer and less crowded α-synuclein fibrils were formed after high temperature pretreatments.

The fibril morphology of α-synuclein after incubation was examined by TEM at 20,000× (scale bar = 500 nm) and 100,000× (scale bar = 100 nm) magnifications. The pretreated temperatures are indicated.

Figure 3. Secondary structures of α-synuclein retained after temperature pretreatments and were fully reversible upon temperature denaturation.

(A) Conformational changes of α-synuclein at 25 µM after pretreatment in 5 different temperatures, 20 (•), 40 (▴), 60 (▪), 80 (⋄), and 95 (▹) °C, for an hr were examined by far-UV CD spectra at room temperature. The data showed α-synuclein adopted random-coiled like structures without conformational changes after the temperature pretreatments. (B) Thermal melting and cooling of 25 µM α-synuclein treated with 20 (•,○) or 95°C (▸,▹). The temperature was increased from 5 to 100°C (solid symbols) or decreased from 100 to 5°C (empty symbols) with temperature changed at a rate of 1.7°C/min. The CD signals at 222 nm were monitored and the molar ellipticity is plotted. The data showed a completely reversible folding of α-synuclein in response to temperature. No protein loss was found in the experiment.

Figure 4. ¹H-¹⁵N HSQC NMR spectra and NMR dynamics of α-synuclein showed no significant difference in chemical shifts and relaxation rates after the temperature pretreatments.

(A) The ¹H-¹⁵N HSQC spectra of 200 µM ¹⁵N-labeled α-synuclein in 10 mM phosphate buffer, pH 7.4 after 1 hr pretreatment at 20 or 95°C. All spectra were obtained at 10°C. (B) R2 relaxation rate of α-synuclein with (○) and without (•) pretreatment at 80°C for 6 hr. All spectra were obtained at 10°C.

Figure 5. Transient oligomers existed in the freshly prepared α-synuclein.

α-Synuclein after pretreatment from 5 different temperatures for an hr were subjected with and without PICUP and western blotting using anti-synuclein antibody, anti-syn211. Metastable oligomers including dimers, trimers, tetramers were observed after PICUP and the amount of oligomers were reduced after higher temperature pretreatments.

Figure 6. The preexisted α-synuclein oligomers were disrupted after 95°C treatment.

α-Synuclein with (dash line) and without (solid line) 95°C treatment were analyzed by AUC-SV and the c(S) distribution is plotted against the sedimentation coefficient ranging from 0 to 7 S. The inset is the enlarged region from c(S) signal below 0.1.

Figure 7. C-terminal truncation was induced after high temperature treatment.

(A) α-Synuclein after treatment at 20 (solid line) and 95°C (dash line) for 1 hr were subjected to analytical SEC. The signal was monitored at absorbance 280 nm. The molecular weight markers are indicated. (B) The truncated species observed after heat treatment was characterized to have a molecular mass of 12,004 kDa by MALDI-TOF mass spectrometry. (C) The sequence identified from LC/MS/MS of the truncated species matched to the N-terminal and NAC regions of α-synuclein. The detected residues are labeled in bold and the arrow heads indicate the predicted truncation sites. The NAC region is boxed.

Figure 8. C-terminal truncated α-synuclein does not undergo fibrillization.

Fibrillization assay of the isolated full-length (•) and C-terminal truncated (▪) α-synuclein. The fibrillization process was monitored by ThT fluorescence.