A protein polymerization cascade mediates toxicity of non-pathological human huntingtin in yeast

Abstract

Several neurodegenerative amyloidoses, including Huntington disease, are caused by expansion of polyglutamine (polyQ) stretches in otherwise unrelated proteins. In a yeast model, an N-terminal fragment of mutant huntingtin with a stretch of 103 glutamine residues aggregates and causes toxicity, while its non-toxic wild type variant with a sequence of 25 glutamines (Htt25Q) does not aggregate. Here, we observed that non-toxic polymers of various proteins with glutamine-rich domains could seed polymerization of Htt25Q, which caused toxicity by seeding polymerization of the glutamine/asparagine-rich Sup35 protein thus depleting the soluble pools of this protein and its interacting partner, Sup45. Importantly, only polymers of Htt25Q, but not of the initial benign polymers, induced Sup35 polymerization, indicating an intermediary role of Htt25Q in cross-seeding Sup35 polymerization. These data provide a novel insight into interactions between amyloidogenic proteins and suggest a possible role for these interactions in the pathogenesis of Huntington and other polyQ diseases.

Figure 1. Toxicity of Htt25Q-GFP polymers correlates with their ability to seed Sup35 polymerization.

(a) Induction of Htt25Q-GFP toxicity by co-production with polyQ/QX-HA proteins. The 74D-694 [psi⁻][PIN⁺] transformants each carrying a plasmid pair of which one plasmid expressed Htt25Q-GFP and another the indicated polyQ/QX-HA protein (-, an empty vector), were grown at 30^oC in liquid SC-Ura-Leu medium with glucose, resuspended in the same medium but with raffinose instead of glucose and after 12 h incubation cell suspensions, diluted to an D600 of 1.0, were spotted onto SC-Ura-Leu plates with galactose as a sole carbon source (Gal) and incubated for 4 days. Equal spotting was controlled by parallel spotting the cells onto same selective plates containing glucose as carbon source (Glu). Four serial 5-fold dilutions of cell suspensions are shown. (b) PolyQ/QX-HA-dependent polymerization of Htt25Q-GFP and Sup35. 74-D694 [psi⁻][PIN⁺] transformants carrying plasmid pairs of which one plasmid either expressed Htt25Q-GFP (+) or did not express this protein (-) and another expressed the indicated polyQ/QX-HA protein, were grown as described in Methods. After incubation in SC-Ura-Leu Gal medium for 10 h, cells were harvested, lyzed and analyzed by SDD-AGE for the presence of SDS-insoluble polymers of polyQ/QX-HA, Htt25Q-GFP and Sup35. Anti-HA, -GFP and -Sup35C antibodies were used.

Figure 2. FCS analysis of HttQ25-RFP aggregation in lysates of cells co-expressing 120QY-HA.

(a) Autocorrelation functions of HttQ25-RFP (black curve) and HttQ25-RFP+120QY-HA (red curve) samples. Green curves are best fit curves using equation (1) (See Methods) with the τ_D = 390 μs (HttQ25-RFP) and τ_D = 1600 μs (HttQ25-RFP+120QY-HA). Grey curves are best fit curves using equation (2) with the τ_D = 615 μs (HttQ25-RFP) and τ_D = 3740 μs (HttQ25-RFP+120QY-HA), and the τ_R = 39 μs (HttQ25-RFP) and τ_R = 43 μs (HttQ25-RFP+120QY-HA). (b) Autocorrelation functions of HttQ25-RFP (black curve), HttQ25-RFP+120QY-HA (red curve), and HttQ103-RFP (blue curve) samples measured under stirring conditions. HttQ103-RFP is shown for comparison.

Figure 3. Comparison of the amount of Htt25Q-GFP and polyQY-GFP polymers.

Transformants of the strain 74-D694 [psi⁻][PIN⁺] with plasmids expressing Htt25Q-GFP in combination with either 76QY-GFP or 120QY-GFP were grown as described in Methods. After incubation in SC-Ura-Leu Gal medium for 10 h, cells were harvested and cell lysates obtained. Then, cell lysates were loaded onto gels without boiling and run for half the length of the gel. The whole gel assembly was then boiled, and the electrophoretic separation was continued. The gels were blotted, and the blots were stained with anti-GFP antibody. Monomer, monomeric form of proteins; polymer, proteins derived from polymers after their dissolution by boiling. Abundances of polyQY-GFP and Htt25Q-GFP polymers were calculated after densitometry of blots using ImageJ software. Six independently-obtained transformants were analyzed and one of the images is presented. Relative polymer abundances were expressed as the means ± standard deviations. The amount of Htt25Q-GFP polymers exceeded that of 76QY-GFP and 120QY-GFP polymers by 1.4 ± 0.2 and 2.0 ± 0.3 fold, respectively.

Figure 4. Co-production of 120QY-HA and Htt25Q-GFP causes toxicity which depends on depletion of soluble Sup35 and Sup45.

(a) Growth of the 74-D694 [psi⁻][PIN⁺] derivatives expressing either 120QY-HA and Htt25Q-GFP, or Htt25Q-GFP alone and carrying plasmids with SUP35, SUP35-C and SUP45, as indicated above the panels. SUP35, the strain with chromosomal wild type SUP35; SUP35∆+plasmid SUP35-C, the strain disrupted for SUP35, which carries centromeric plasmid with SUP35-C encoding Sup35 devoid of the prionogenic NM region; SUP35+plasmid SUP35-C, the strain with chromosomal wild type SUP35 which carries centromeric plasmid with SUP35-C; SUP35+plasmid SUP45, the strain with chromosomal wild type SUP35, which carries centromeric plasmid with SUP45; SUP35+plasmid SUP35-C SUP45, the strain with chromosomal wild type SUP35, which carries centromeric plasmid with SUP35-C and SUP45. Four serial 5-fold dilutions of cell suspensions are shown. For other details, see legend to Fig. 1. (b) Centrifugation analysis of levels of soluble Sup35 and Sup45. Cell lysates were fractionated by ultracentrifugation and supernatants were analyzed by SDS-PAGE and Western blotting. Staining with either anti-Sup35NM (Sup35) or anti-Sup45 (Sup45) antibodies. Relative abundances of soluble Sup35 and Sup45 in transformants expressing HttQ103-GFP, HttQ25-GFP in combination with 120QY-HA or non-toxic and non-aggregating HttQ25-GFP alone, used as a control, were calculated after densitometry of blots using ImageJ software. Three independently-obtained transformants of each type were analyzed and the relative protein abundances presented on histograms are expressed as the means ± standard deviations. A typical blot image is presented.

Figure 5. Htt25Q-GFP aggregates and causes toxicity in [PIN⁺] cells overexpressing Rnq1.

(a) Growth of the 74-D694 [psi⁻][PIN⁺] strain which carries multicopy plasmids encoding either Rnq1 or Htt25Q-GFP, or both proteins simultaneously, was analyzed as described in legend to Fig. 1. Four serial 5-fold dilutions of cell suspensions are shown. (b) Polymerization of Sup35 depends on polymers of Htt25Q-GFP generated in [PIN⁺] cells upon overproduction Rnq1. Polymers of Rnq1, Htt25Q-GFP and Sup35 proteins in lysate of 74-D694 [psi⁻][PIN⁺] were visualized by SDD-AGE followed by Western blotting with the use anti-GFP, anti-Rnq1 and anti-Sup35 polyclonal antibodies.

Figure 6. Toxicity of Htt25Q-GFP in [PSI⁺] cells is not related to its polymerization.

(a) Growth of the 74-D694 [PSI⁺][PIN⁺] strain and its [psi⁻][PIN⁺] derivative carrying either a multicopy plasmid encoding Htt25Q-GFP or empty vector (−). Where indicated, transformants in addition carry either centromeric plasmid encoding Sup35C, or empty vector (−). Growth of transformants was analyzed as described in legend to Fig. 1. Four serial 5-fold dilutions of cell suspensions are shown. (b) Htt25Q-GFP does not form polymers both in [PSI⁺][PIN⁺] in [psi⁻][PIN⁺] cells of 74-D694. The transformant simultaneously expressing Htt25Q-GFP and 120QY-GFP was used as a positive control for polymer formation. Polymers of Htt25Q-GFP were visualized by SDD-AGE followed by Western blotting and staining with anti-GFP antibody.

Figure 7. Intermediary cross-seeding and a protein polymerization cascade.