fibrils, however, oligomeric structures and amorphous protein aggregates were

Transcription

1 Supplementary Figure 1: Effect of equimolar EGCG on αs and Aβ aggregate formation. A D B E αs C F Figure S1: (A-C) Analysis of EGCG treated αs (1 µm) aggregation reactions by EM. A 1:1 molar ratio of EGCG relative to αs was added to the polymerization reactions. αs samples were analyzed after 16 h. (D-F) Analysis of EGCG treated Aβ42 (15 µm) aggregation reactions by EM after 72 h incubation in SP buffer. EGCG was added at a molar ratio of 1:1 relative to Aβ42. In the presence of EGCG, the formation of typical amyloid fibrils was prevented for both αs and Aβ42. Instead of fibrils, however, oligomeric structures and amorphous protein aggregates were observed; all scale bars 1 nm.

2 Supplementary Figure 2: EGCG stimulates the formation of SDS-stable oligomers B EGCG 1x 5x 1x control C control D EGCG 1x 5x 1x A [h] αs, silver stain αs, NBT stain m silver stain NBT stain Figure S2: Analysis of EGCG-induced formation of αs SDS-resistant oligomers. αs (1 µm) was incubated with or without EGCG (1 µm 1 mm) at 37 C for 2 h and samples were analyzed by SDS-PAGE and silver staining (A), or blotted onto nitrocellulose membrane for NBT staining (B). Time resolved analysis of untreated αs protein. αs (1 µm) was incubated without EGCG at 37 C for the indicated times and analyzed by SDS-PAGE and silver staining (C). In addition, the protein was blotted onto membranes and examined by NBT staining (D); m indicates monomer bands. Our data clearly show that an equimolar concentration of EGCG relative to αs is sufficient to stimulate the assembly of SDS-stable oligomers. No such structures are formed in the absence of chemical compound. [h]

3 Supplementary Figure 3: EGCG directly binds to natively unfolded αs protein Figure S3: The indicated proteins (2 µm) were incubated for 2 h at 37 C with EGCG (2 µm) in TBS buffer, separated by SDS-PAGE and electroblotted onto nitrocellulose membranes. SDS gels were stained with silver; nitrocellulose membranes with NBT 4. We found that EGCG binds to unstructured αs but not to the folded control proteins ovalbumin, carbonic anhydrase, chymotrypsinogen or lysozyme. *, indicates NBT staining of αs.

4 Supplementary Figure 4: An equimolar concentration of EGCG relative to αs inhibits the formation of A11-positive amyloid oligomers A B EGCG control h 1 h 2 h 4 h 6 h A11 dot blot signal [arb.] x EGCG no EGCG 8h 1 h 1h 2h 4h 6h 8h incubation time Figure S4: Detection of A11-reactive amyloid oligomers in unseeded αs (1 µm) polymerization reactions with (1 µm) or without EGCG. At various times, aliquots were applied to nitrocellulose and probed with oligomer-specific antibody A11. (A) A11 dot blot. (B) Quantitative analysis of A11 blot signals (n=3). αs was pretreated by size exclusion chromatography to remove preformed αs oligomers, which are normally present in purified protein solutions (Fig. 4B). The compound very efficiently prevented the formation of A11-reactive oligomers that appear after an incubation period of 2 h in untreated aggregation reactions.

5 Supplementary Figure 5: Generation of fibrillar αs aggregates and EGCG-induced spherical αs oligomers for MTT metabolic assays. Figure S5: EGCG oligomers were produced by incubating αs protein (1 µm) with a 5-fold molar excess of chemical compound for 24 h at 37 C. αs fibrils were produced under the same conditions without EGCG.

6 Supplementary Figure 6: Cytotoxicity of αs and Aβ42 monomers in cell-based assays A 14 MTT reducing activity (%) fibrils monomer control αs concentration (nm) B MTT reducing activity (%) 1% 5% % buffer fibril monomer Aβ42 concentration (nm) Figure S6: Cytotoxic effects of αs and Aβ 42 fibrils and monomers on PC12 cells. (A) αs fibrils but not monomers are toxic for PC12 cells. αs monomers are even protective for PC12 cells, as has been described previously in the literature 5. (B) Cytotoxic effect of β-sheet-rich fibrils and Aβ 42 monomers on PC12 cells. Monomeric Aβ 42 is non-toxic for PC12 cells. Values represent means ± SD (n = 3).

7 Supplementary Figure 7: Equimolar concentrations of EGCG relative to Aβ 42 induce the formation of SDS-stable oligomers. Control 24 EGCG : Aβ42 1: Incubation time [h] Figure S7: Aβ 42 (15 µm) was incubated with EGCG at a 1:1 molar ratio in SP-buffer at 37 C. Subsequently, samples were analyzed by SDS-PAGE and Western blotting (mab 6E1). The first SDS-stable Aβ 42 oligomers were observed after an incubation period of 5 h. In contrast, no such structures were observed in the absence of EGCG. (control).

8 Supplementary Information Supplementary Methods Production and purification of recombinant αs protein. Alpha-synuclein (αs) cdna (WT) was obtained from plasmid ppc97 1 and subcloned as a SalI-NotI fragment into the expression plasmid pqe32n 2 yielding pqe32n-αswt. The E. coli strain SCS1/pSE111 3 was transformed with pqe32n-αswt, and clones producing recombinant protein upon induction with isopropyl-d-thiogalactopyranoside (IPTG) were identified. The correct nucleotide sequence of the insert was confirmed by DNA sequencing. Recombinant His-tagged αs protein was purified under denaturing conditions with 8 M urea using a Ni-NTA affinity resin (Qiagen) according to manufacturer s instructions. After purification, the urea-containing buffer was exchanged for TBS via a PD1 desalting column (Amersham Pharmacia). Purified protein solutions were kept on ice for immediate use.

9 Supplementary References 1. Engelender, S. et al. Synphilin-1 associates with alpha-synuclein and promotes the formation of cytosolic inclusions. Nat Genet 22, (1999). 2. Wanker, E. E. et al. HIP-I: a huntingtin interacting protein isolated by the yeast two-hybrid system. Hum Mol Genet 6, (1997). 3. Bussow, K. et al. A method for global protein expression and antibody screening on high-density filters of an arrayed cdna library. Nucleic Acids Res 26, (1998). 4. Paz, M. A., Fluckiger, R., Boak, A., Kagan, H. M. & Gallop, P. M. Specific detection of quinoproteins by redox-cycling staining. J Biol Chem 266, (1991). 5. El-Agnaf, O. M. et al. Aggregates from mutant and wild-type alpha-synuclein proteins and NAC peptide induce apoptotic cell death in human neuroblastoma cells by formation of beta-sheet and amyloid-like filaments. FEBS Lett 44, 71-5 (1998).