Ubiquitin-interacting motifs of ataxin-3 regulate its polyglutamine toxicity through Hsc70-4-dependent aggregation
- Department of Pharmacology, Wayne State University, United States
- Department of Neurology, Wayne State University, United States
Figures
Figure 1 with 1 supplement
Ataxin-3 domains impact its polyQ toxicity.
(A) Diagram of ataxin-3. Josephin: deubiquitinase domain necessary for ataxin-3's ability to cleave ubiquitin bonds (Winborn et al., 2008). UbS: ubiquitin-binding site (Nicastro et al., 2010; Nicastr…
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Figure 1—source data 1
Data related to Figure 1 - Longevity and motility results.
- https://cdn.elifesciences.org/articles/60742/elife-60742-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Ataxin-3 domains impact the toxicity of its polyQ-expanded variant in glia.
(A) Longevity curves from adults expressing the noted versions of ataxin-3 in all fly glia, throughout development and in adults. These are the same lines as in Figure 1. P values are from log-rank …
Figure 2 with 1 supplement
UIMs modulate toxicity of the isolated polyQ of ataxin-3.
(A) Diagram (top) and abbreviations (bottom) for transgenes used. The polyQ of ataxin-3 has a lysine interruption after the first three glutamines (QQQKQQQQ…), highlighted in yellow. (B) Summary of …
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Figure 2—source data 1
Data related to Figure 2 - Longevity results.
- https://cdn.elifesciences.org/articles/60742/elife-60742-fig2-data1-v2.xlsx
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Figure 2—source data 2
Data related to Figure 2 - Uncropped images.
- https://cdn.elifesciences.org/articles/60742/elife-60742-fig2-data2-v2.pdf.zip
Figure 2—figure supplement 1
Levels of polyQ proteins, related to Figure 2D.
Western blots from lysates of whole, dissected fly heads; 25 heads per group. We caution here that the comparison of overall polyQ proteins is not precise, especially concerning polyQ80-UIM3, which …
Figure 3
Mutating the UIMs of full-length, pathogenic ataxin-3 decreases its toxicity.
(A) Amino acid sequence of full-length, pathogenic ataxin-3 and UIM mutations. (B) Summary of results when ataxin-3 flies, or non-expressing controls, are monitored during development and in …
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Figure 3—source data 1
Data related to Figure 3 - Longevity and motility results.
- https://cdn.elifesciences.org/articles/60742/elife-60742-fig3-data1-v2.xlsx
Figure 4
Mutating the UIMs of pathogenic ataxin-3 decreases its aggregation.
(A) Western blots from simple lysates of adults expressing transgenes pan-neuronally. Red arrows: likely proteolytic products of ataxin-3 that we observe sometimes. (B) Cytoplasmic/nuclear …
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Figure 4—source data 1
Data related to Figure 4 - Numerical data.
- https://cdn.elifesciences.org/articles/60742/elife-60742-fig4-data1-v2.xlsx
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Figure 4—source data 2
Data related to Figure 4 - Uncropped images.
- https://cdn.elifesciences.org/articles/60742/elife-60742-fig4-data2-v2.pdf.zip
Figure 5
Pathogenic ataxin-3 interacts with Hsc70-4 in a UIM-dependent manner.
(A) SDS-PAGE gel stained for all proteins with Sypro Ruby. Boxed areas were excised and proteins were identified with LC/MS/MS. Two versions of immunopurifications (IPs) were conducted. Middle panel …
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Figure 5—source data 1
Data related to Figure 5 - Uncropped images.
- https://cdn.elifesciences.org/articles/60742/elife-60742-fig5-data1-v2.pdf.zip
Figure 6 with 1 supplement
Hsc70-4 knockdown improves ataxin-3 toxicity in fly eyes.
(A) Western blots from dissected fly heads. Histograms are from blots on top and other, independent biological repeats. Shown are means -/+ SD. Statistics: one-way ANOVA with Dunnett's correction. …
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Figure 6—source data 1
Data related to Figure 6 - Numerical data.
- https://cdn.elifesciences.org/articles/60742/elife-60742-fig6-data1-v2.xlsx
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Figure 6—source data 2
Data related to Figure 6 - Uncropped images.
- https://cdn.elifesciences.org/articles/60742/elife-60742-fig6-data2-v2.pdf.zip
Figure 6—figure supplement 1
Uncropped blot from panel 6E.
Figure 7
Hsc70-4 knockdown improves toxicity from independent, truncated polyQ models.
Representative images of fly eyes (A, C) and fly eye sections (C) with the noted genotypes. Photos are representative of results from three independent sets of crosses. (B) Western blots from …
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Figure 7—source data 1
Data related to Figure 7 - Uncropped images.
- https://cdn.elifesciences.org/articles/60742/elife-60742-fig7-data1-v2.pdf.zip
Figure 8
UIMs are important for inter-ataxin-3 interactions.
(A, B) Western blots from IPs of ataxin-3 with expanded (A) or wild-type (B) polyQ repeats expressed in fly eyes. All transgenes (HA- or V5-tagged) were expressed at the same time and in the same …
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Figure 8—source data 1
Data related to Figure 8 - Uncropped images.
- https://cdn.elifesciences.org/articles/60742/elife-60742-fig8-data1-v2.pdf.zip
Figure 9 with 1 supplement
Proposed model.
(A) Ataxin-3 UIMs enable its interaction with Hsc70-4 and other ataxin-3 proteins, both of which enhance aggregation and toxicity. UIM3 (thicker lines) appears to be a stronger contributor to these …
Figure 9—figure supplement 1
Relative importance of UIMs on fly longevity.
(A) Longevity curves of adult flies expressing the denoted transgenes pan-neuronally. Continuous curves are the same as in Figure 1C; X-axis is extended to 86 days to show the full longevity of …
