Degron capability of the hydrophobic C-terminus of the polyglutamine disease protein, ataxin-3

Abstract

Ataxin-3 is a deubiquitinase and polyglutamine disease protein whose cellular properties and functions are not entirely understood. Mutations in ataxin-3 cause spinocerebellar ataxia type 3 (SCA3), a neurodegenerative disorder that is a member of the polyglutamine family of diseases. Two major isoforms arise from alternative splicing of ATXN3 and are differently toxic in vivo as a result of faster proteasomal degradation of one isoform compared to the other. The isoforms vary only at their C-termini, suggesting that the hydrophobic C-terminus of the more quickly degraded form of ataxin-3 (here referred to as isoform 2) functions as a degron-that is, a peptide sequence that expedites the degradation of its host protein. We explored this notion in this study and present evidence that: (a) the C-terminus of ataxin-3 isoform 2 signals its degradation in a proteasome-dependent manner, (b) this effect from the C-terminus of isoform 2 does not require the ubiquitination of ataxin-3, and (c) the isolated C-terminus of isoform 2 can enhance the degradation of an unrelated protein. According to our data, the C-terminus of ataxin-3 isoform 2 is a degron, increasing overall understanding of the cellular properties of the SCA3 protein.

Keywords: RRID:AB_1281300; RRID:AB_2307391; RRID:SCR_001010; ataxia; deubiquitinase; isoform; neurodegeneration; polyglutamine; proteasome; ubiquitin.

FIGURE 1

Ataxin-3 isoforms. Diagrammatic representation of the human ATXN3 gene and the ataxin-3 protein isoforms that arise from its differential splicing. Iso2_tr: a single-nucleotide polymorphism leads to a truncated version of isoform 2. DUB, deubiquitinase; PolyQ, polyglutamine; UIM, Ubiquitin-Interacting Motif; U, untranslated regions. Sequences below show the amino acid composition of the human ataxin-3 isoforms. Common regions are in gray lettering. The HA tag that is appended in frame at the C-terminal end is shown in green font

FIGURE 2

Isoform 2 of ataxin-3 is present at lower protein levels in mammalian cells. (a–e) Western blots from whole cell lysates of HEK-293T cells transfected with ataxin-3 with a normal polyQ of 12 repeats, as indicated. Where noted, cells were treated overnight with DMSO, 20 μM MG132 (MG; in DMSO) or 100 μM chloroquine (CQ; in DMSO). Graphs in panels a–e are box-and-whisker plots of data quantified from respective images above each graph and other, independent biological replicates. p values for panels b–e are from one-tailed Mann-Whitney U tests comparing MG132- or chloroquine-treated cells to their DMSO-treated counterparts. p values for panel a are two-tailed Mann–Whitney U tests comparing all variants to “Iso1.” (f) Box-and-whisker plots comparing the effect of MG132 treatment on the isoforms of ataxin-3. Data were compiled by graphing values from MG132-treated, HEK-293T cells expressing the denoted isoforms, normalized to their respective, DMSO-treated cells and expressed as percent change. p values are from two-tailed studenťs t-tests comparing isoform 2 to isoform 1 from data in panels b–e and other, independent biological replicates. (g) qRT-PCR data measuring the noted mRNA levels compared statistically to isoform 1. p values are from two-tailed Mann–Whitney U tests. (h) Western blots from HEK-293T cells transfected with ataxin-3 constructs harboring an expanded polyQ of 80 repeats. Lanes are from the same blot and same exposure, cropped and rearranged for ease of viewing. In panels a and h: black arrows denote the main, unmodified band of ataxin-3 protein and purple arrows denote ubiquitinated species of ataxin-3 that we sometimes, but not always, observe in this type of assay. In all graphs, “N”s denote independent biological replicates

FIGURE 3

The tail of isoform 2 of ataxin-3 reduces eGFP stability in mammalian cells. (a) Construct design. Underlined amino acids in black font result from cloning. (b) Steady-state levels of eGFP variants transfected into HEK-293T cells and harvested 24 hr later. Whole cell lysates. Graph is from images on the left and other, independent biological replicates. p value is from two-tailed Mann–Whitney U test. (c) Whole cell lysates of HEK-293T cells transfected as noted and treated with cycloheximide (CHX; 100 μM) 24 hr later for the indicated amounts of time. Images are from the same membrane, exposed for different amounts of time to approximate t=0 hr levels of the two constructs. Graph below is from quantification of signal from images above and other, independent biological repeats. p values are from studenťs t-tests comparing levels at each respective point for “eGFP+Iso2 Tail” to “eGFP.” Similar results were obtained from ANOVA with Tukey’s post hoc. (d–f) Steady-state levels of eGFP variants from whole cell lysates. HEK-293T cells were transfected as indicated and 24 hr later were treated overnight with MG132 (20 μM) or chloroquine (CQ; 100 μM) dissolved in DMSO, or with DMSO alone. Black arrow in panel d: main eGFP band. Gray arrow in panel d: another anti-GFP-positive band that we observe sometimes, but not always, that might be a proteolytic fragment of full eGFP. Graphs in panels d–f are from respective images on top and additional biological repeats. p values are from one-tailed Mann–Whitney U tests comparing MG132- or chloroquine-treated cells to DMSO-treated counterparts. For panels b–f: Note that “eGFP+Iso2 Tail” migrates more quickly on SDS-PAGE gels than “eGFP,” similar to what we observed before with ataxin-3 isoform 2 in flies (Johnson et al., 2019) and in mammlian cells ((Johnson et al., 2019); Figure 2). This pattern most likely results from the hydrophobicity of the tail of isoform 2, which leads host proteins to migrate not quite as expected on SDS-PAGE gels. As with all other plasmids used in this work, plasmid identity and integrity was confirmed with site-restricted digests and two rounds of sequencing, which confirmed that “eGFP+Iso2 Tail” was intact. (g) qRT-PCR results comparing the levels of “eGFP” to “eGFP+Iso 2 Tail.” p value is from two-tailed Mann–Whitney U test. “N”s in all graphs denote independent biological replicates

FIGURE 4

The tail of isoform 2 of ataxin-3 does not align with non-ataxin-3 proteins. Summary of findings from BLASTp analysis of the sequence shown in the figure. No specific organism was entered at the input stage. The amino acid sequence queried was blasted against all organisms. The species shown are the only ones that emerged from this analysis

FIGURE 5

Folding differences between isoforms 1 and 2 of ataxin-3. Shown are the results from three different folding prediction software programs (Materials and Methods) comparing the full-length sequences of isoforms 1 and 2 of wild-type ataxin-3. Y-axes indicate predicted order/disorder. Vertical dashed lines demarcate the tails of isoforms 1 and 2