Essential role of coiled coils for aggregation and activity of Q/N-rich prions and PolyQ proteins

Abstract

The functional switch of glutamine/asparagine (Q/N)-rich prions and the neurotoxicity of polyQ-expanded proteins involve complex aggregation-prone structural transitions, commonly presumed to be forming β sheets. By analyzing sequences of interaction partners of these proteins, we discovered a recurrent presence of coiled-coil domains both in the partners and in segments that flank or overlap Q/N-rich and polyQ domains. Since coiled coils can mediate protein interactions and multimerization, we studied their possible involvement in Q/N-rich and polyQ aggregations. Using circular dichroism and chemical crosslinking, we found that Q/N-rich and polyQ peptides form α-helical coiled coils in vitro and assemble into multimers. Using structure-guided mutagenesis, we found that coiled-coil domains modulate in vivo properties of two Q/N-rich prions and polyQ-expanded huntingtin. Mutations that disrupt coiled coils impair aggregation and activity, whereas mutations that enhance coiled-coil propensity promote aggregation. These findings support a coiled-coil model for the functional switch of Q/N-rich prions and for the pathogenesis of polyQ-expansion diseases.

Figure 1. Overrepresentation of CCs in Q/N-rich and polyQ proteins and in their interactomes

A. Per-residue CC probability for Hsp104, CHIP, PQBP, and HIP-1 (from 0 to 1) obtained with Coils. Peaks with the highest probability (0.8-1) are highlighted in black. B. Proportion of CC proteins among interactors of Ure2, apCPEB, and Htt, and among known and candidate yeast prions (Alberti et al., 2009), and human polyQ-expansion proteins, as compared with eukaryotic proteomes. C. CC probability for prions, and amyloids, compared with proteins with known CC structure, obtained with Paircoil2 and Coils. Red bars represent Q/N-rich (>15%) or polyQ regions. See also Fig. S1.

Figure 2. Heptad repeats in Q/N-rich and polyQ proteins

A. Scheme of two coiled alpha-helices. B and C. Lateral and zenithal view of two coiled helices. Red circles= heptad positions a/d, green circles= g/e, and cyan-to-blue circles=b, c and f. D. Modified helical net representation of the sequences shown in A. Note the vertical alignment of a/d residues, and the grouping of a/d hydrophobic residues in discrete clusters along the helices. Asterisks indicate stutters. See also Fig. S2.

Figure 3. Structure-guided mutagenesis of Ure,2 apCPEB, and Htt-72Q N-terminal domains

A. Aminoacidic substitutions in each mutant of Ure2, apCPEB, Htt-25Q, Htt-75Q, and Aβ(1-42). Mutants are grouped as CC-disrupting (cc-), CC-enhancing (cc+), CC-neutral (cc0), and β-sheet-breaking (β-). B. CC propensity of mutant proteins shown in A. See also Fig. S3.

Figure 4. Secondary structure and oligomeric state of Q/N-rich and polyQ peptides

A. Primary sequence of the peptides used for CD experiments. A C-terminal YK di-peptide was added to those peptides devoid of Y/W residues to allow the spectrophotometic determination of concentration (Y) and improve solubility (K). All peptides were N-terminally acetylated and C-terminally amidated. B,D,F,H. CD spectra of the peptides shown in A under different temperature conditions (see text). C, E, G, I. Quantification of the [θ] 222/208 ratio for the indicated peptides as a function of TFE concentration or temperature. L, M, N. Silver-stained tricine-SDS-PAGE of the indicated peptides after glutaraldehyde cross-linking (+) at 37 °C for 15 min. Arrowheads indicate monomeric (lower) and dimeric (higher) species. Asterisks indicate supradimeric species. See also Fig. S4 and Table S1.

Figure 5. CC disruption impairs aggregation of Ure2, apCPEB, and Htt72Q

A. Phase contrast (1,4) and fluorescence micrographs of ure2Δ yeast cells overexpressing Ure2-GFP (2,3) or Ure2/cc-/-GFP (5,6), at low and high magnification. Arrowheads indicate aggregates, arrows indicate cells with diffuse fluorescence. Cal.: 20 μm. B. Phase contrast (1,5) and fluorescence micrographs of Aplysia neurons overexpressing apCPEB-GFP (1-4) or apCPEB/cc-/#2-GFP (5-8), at low and high magnification. Arrowheads and arrows as in A. Cal.: 50 μm. C. Fluorescence micrographs of HEK293 cells overexpressing either Htt-72Q-GFP, or its cc- mutants /#1, /#2, and /W. Arrowheads and arrows as in A. Cal.: 20 μm. D. Phase contrast and fluorescence micrographs of Aplysia neurons overexpressing either Htt-72Q (1,2) or Htt-72Q/cc-/#2 (3,4) for 72h. Cal.: 75 μm. See also Fig. S5.

Figure 6. Aggregation of CC-enhancing and CC-neutral mutants

A, B. Confocal images of HEK293 cells overexpressing for 72 hours Htt-25Q, Htt-72Q, and their cc+ or cc0 mutants. Arrowheads indicate aggregates, arrows diffuse fluorescence. Cal.: 30 μm C. HEK293 cells overexpressing for 72 hours either GFP-Aβ(1-42) or its mutant β-/#1. D. Pseudocolor images of aggregates and fibers formed by Htt-72Q and cc0 mutants. Cal.: 10 μm. E, F, G. Histograms (mean ± SEM) showing the percentage of aggregate-containing cells on the total number of cells overexpressing wt and mutant forms of Ure2, Htt, and Aβ(1-42). See also Fig. S6.

Figure 7. CC propensity regulates insolubility and protein activity/toxicity

A, C, E. Western blots of ultracentrifugation assays on lysates of yeast (A) or HEK293 cells (C,E) overexpressing wt and CC-mutant Ure2, Htt, and apCPEB. Soluble (S) and insoluble pellet (P) fractions are shown. B, D, F. Relative proportion of S and P fractions of wt and CC mutant forms of Ure2, Htt, and apCPEB in ultracentrifugation assays (mean ± SEM). G. Photographs of ure2Δ S. cerevisiae transformants overexpressing either wt or cc- mutant Ure2, plated in serial dilutions on a substrate containing either uracil or USA. H. Number of USA+ colonies formed (at 10^-3-10^-5 dilutions) by ure2Δ S. cerevisiae transformants overexpressing wt or cc- Ure2-GFP. Data (mean ± SEM) are normalized to the average number of colonies formed by Ure2-GFP transformants at each dilution. I. Relative toxicity of Htt-72Q and of its CC mutants after 72 hours of overexpression, as assayed with the MTT-formazan assay. J Schematic representation of the possible role of CCs in the conformational dynamics of Q/N-rich and polyQ proteins. Alpha-helices/CCs may represent intermediate structures facilitating the misfolding of helical protomers (red arrowhead) or CC fibers (red arrow) into β-sheet polymers (upper panel), or self-sufficient mediators of conformational change and aggregation (middle and lower panel). See also Fig. S7.