Characterization of Amyloid Cores in Prion Domains

Abstract

Amyloids consist of repetitions of a specific polypeptide chain in a regular cross-β-sheet conformation. Amyloid propensity is largely determined by the protein sequence, the aggregation process being nucleated by specific and short segments. Prions are special amyloids that become self-perpetuating after aggregation. Prions are responsible for neuropathology in mammals, but they can also be functional, as in yeast prions. The conversion of these last proteins to the prion state is driven by prion forming domains (PFDs), which are generally large, intrinsically disordered, enriched in glutamines/asparagines and depleted in hydrophobic residues. The self-assembly of PFDs has been thought to rely mostly on their particular amino acid composition, rather than on their sequence. Instead, we have recently proposed that specific amyloid-prone sequences within PFDs might be key to their prion behaviour. Here, we demonstrate experimentally the existence of these amyloid stretches inside the PFDs of the canonical Sup35, Swi1, Mot3 and Ure2 prions. These sequences self-assemble efficiently into highly ordered amyloid fibrils, that are functionally competent, being able to promote the PFD amyloid conversion in vitro and in vivo. Computational analyses indicate that these kind of amyloid stretches may act as typical nucleating signals in a number of different prion domains.

Figure 1. The four proteins studied here are represented with their respective known functional domains (lilac).

Swi1: (1) ARID/BRIGHT DNA binding domain; Sup35: (1) Elongation factor Tu GTP binding domain, (2) Elongation factor Tu domain 2 and (3) Elongation factor Tu C-terminal domain; Ure2: (1) Glutathione S-transferase N-terminal domain and (2) Glutathione S-transferase C-terminal domain. Prion domains are shown in green and the predicted 21 residues amyloid core inside the prion domain in red. The functional domains were assigned according to Pfam (http://pfam.xfam.org) and the PFD according to UniProt (http://uniprot.org). The exact position of the amyloid cores in the PFDs and their amino acid sequences are displayed in Table 1.

Figure 2. PDFs amyloid cores secondary structure.

The peptides secondary structure was analyzed by Far-UV CD (a) before (solid lines) and after incubation (dotted lines). The initial characteristic 200 nm negative peak corresponding to disordered structure displaces to 216 nm, indicative of a β-sheet content, upon incubation. The secondary structure of the incubated peptides was also analysed by ATR-FTIR (b). All of them exhibit a major band at ∼1625 cm⁻¹ indicating the presence of inter-molecular β-sheets. Additional secondary structure components are detailed in Table 2.

Figure 3. Thioflavin-T binding to PDFs amyloid cores.

The peptides were let to aggregate in quiescent solutions for 2 hours at RT and 100 μM. Then, 10 μL of soluble or aggregated samples were mixed with a solution of Th-T at 30 μM and fluorescence was measured by exciting the samples at 450 nm and collecting their emission from 460 to 600 nm.

Figure 4. Fibrillar structure of Sup35, Mot3, Swi1 and Ure2 PFDs amyloid cores.

On the upper images bars corresponds to 1 μm and on the lower images to 0.5 μm for Sup35, Mot3 and Ure2 and to 0.2 μm for Swi1.

Figure 5. X-ray diffraction patterns collected from partially aligned fibril samples formed by the PFDs.

Fiber axis is vertical and the arrows highlight the characteristic “cross-β” reflections. Grey 4.76 Å meridional, white 9–11 Å equatorial reflections.

Figure 6. Aggregation kinetics, seeding and cross-seeding reactions of Sup35 PFD amyloid core and Sup35-NM domain.

In panel a the aggregation kinetics were carried out by incubating 100 μM of peptide with 30 μM of Th-T at RT with no agitation and the fluorescence measured along time. Th-T spectra were collected by exciting the samples at 450 nm and collecting the emission from 460 to 600 nm. The intensity at 482 nm was used to monitor the extent of aggregation. In the inset are shown some representative spectra. In panel b, the Sup35-NM domain was incubated at 10 μM, at RT, under agitation of 400 rpm in the absence of seeds or self-seeded with 2% of its own fibrils or seeded with 2% of the fibrils formed by the Sup35 PFD peptide. The three reactions were carried out simultaneously and monitored by Th-T fluorescence. At each time point, a 10 μL aliquot of each sample was mixed with 30 μM of Th-T solution and the fluorescence spectra collected. The intensity at 482 nm was used as a proxy of fibril formation.

Figure 7. Conversion of non prionic [psi⁻] to prionic [PSI⁺] yeasts by transformation with Sup35 PFD amyloid core peptide or the complete Sup35-NM domain.

Yeast cells (L1749) were transformed by incubation of spheroplasted cells with the transformation mixtures, containing either 10 μg/mL pRS416 vector, or the pRS416 vector plus 40 μM of Sup35 peptide fibrils, or pRS416 plus 40 μM of NM-domain fibrils. 100 colonies were plated and screened for PSI positive and negative prion phenotypes in ¼ YPD plates. Representative positive and negative colonies are shown in this figure, together with the positive and negative control strains: L1749 [psi⁻] and L1762 [PSI⁺].