Poly(Q) Expansions in ATXN7 Affect Solubility but Not Activity of the SAGA Deubiquitinating Module

Abstract

Spinocerebellar ataxia type 7 (SCA7) is a debilitating neurodegenerative disease caused by expansion of a polyglutamine [poly(Q)] tract in ATXN7, a subunit of the deubiquitinase (DUB) module (DUBm) in the SAGA complex. The effects of ATXN7-poly(Q) on DUB activity are not known. To address this important question, we reconstituted the DUBm in vitro with either wild-type ATXN7 or a pathogenic form, ATXN7-92Q NT, with 92 Q residues at the N terminus (NT). We found that both forms of ATXN7 greatly enhance DUB activity but that ATXN7-92Q NT is largely insoluble unless it is incorporated into the DUBm. Cooverexpression of DUBm components in human astrocytes also promoted the solubility of ATXN7-92Q, inhibiting its aggregation into nuclear inclusions that sequester DUBm components, leading to global increases in ubiquitinated H2B (H2Bub) levels. Global H2Bub levels were also increased in the cerebellums of mice in a SCA7 mouse model. Our findings indicate that although ATXN7 poly(Q) expansions do not change the enzymatic activity of the DUBm, they likely contribute to SCA7 by initiating aggregates that sequester the DUBm away from its substrates.

FIG 1

Reconstitution of mammalian DUBm. (A) Schematic of the mammalian SAGA DUBm. (B) Schematic representation of ATXN7 N-terminal fragments with 24 Q residues and 92 Q residues, where Q is glutamine. (C) Colloidal staining of DUBm subunits after elution with HA or Flag peptides from immunoaffinity agarose beads. The expression levels are similar for all the subunits analyzed, with the exception of the expression level for ATXN7-92Q NT. #, purified DUBm subunits in each lane. (D) Expression of exogenous Flag-ATXN7 NT in WCEs from Sf21 insect cells after cellular fractionation at 3 days postinfection in order to assess the solubility of ATXN7-24Q NT and ATXN7-92Q NT. Lanes S, supernatant (soluble fraction); lanes P, pellet (insoluble fraction). Numbers on the left are molecular masses (in kilodaltons). (E) Colloidal staining of the reconstituted DUBm with ATXN7-24Q NT or ATXN7-92Q NT after anti-HA (USP22) immunoprecipitation. (F) Immunoblot analysis of the reconstituted DUBm for which the results are presented in panel E. ATXN7-24Q NT or ATXN7-92Q NT, ATXN7L3, and ENY2 are all Flag tagged and are detected with anti-Flag antibody; HA-USP22 is detected with anti-HA antibody. *, modified forms of ATXN7-24Q NT or ATXN7-92Q NT due to ubiquitination (data not shown). Lanes M (C and E), molecular mass markers; IB, immunoblot analysis; IP, immunoprecipitation analysis.

FIG 2

ATXN7-24Q NT promotes robust DUB activity of the DUBm in vitro. (A) In vitro deubiquitination assays using core histones as the substrates were performed with USP22 alone (lanes 2 and 3), trimeric DUBm (no ATXN7-24Q NT; lane 4), and tetrameric DUBm (with ATXN7-24Q NT, lanes 5 and 6). The USP22 C185S catalytically inactive mutant served as a negative control (lanes 3 and 5). The trimeric and tetrameric complexes had similar activities on ubiquitinated H2B after 2 h. (B) In vitro DUB assays using core histones were performed for different combinations of DUBm subunits coinfected in Sf21 insect cells, in order to test potential enzymatic activity. Similar amounts of USP22 in these complexes were incubated with core histones for 2 h. No activity was observed in any dimeric complex (lanes 5 and 6), and the only trimeric complex with obvious enzymatic activity contained USP22, ATXN7L3, and ENY2 (lane 3). (C and D) In vitro DUB assays using core histones were performed with USP22 alone (lanes 1 to 4), DUBm (no ATXN7-24Q NT; lanes 5 to 8), and DUBm (with ATXN7-24Q NT; lanes 9 to 12), and samples were collected for analysis at the indicated time points. H2A (D) and H2B (C) ubiquitination was monitored with antibodies specific for these modifications. In all assays (A to D), core histones were purified from HEK293T cells. (E) Hydrolysis of ubiquitin-AMC (0.5 μM) alone or with similar amounts of recombinant USP22, DUBm (no ATXN7-24Q NT), and DUBm (with ATXN7-24Q NT). The release of AMC fluorescence was monitored by a spectrophotometer at 528 nm. The tetrameric DUBm with ATXN7-24Q NT showed the highest enzymatic activity. The USP22 WT or catalytic mutant was tagged with HA; ATXN7-24Q NT, ATXN7L3, and ENY2 were all tagged with Flag.

FIG 3

ATXN7-92Q NT is not defective in promoting DUBm activity. (A) Purification of the recombinant DUBm using two sequential affinity precipitations with anti-HA– and anti-Flag–agarose beads, respectively. Comparison of the two purified DUBm complexes containing ATXN7-24Q NT or ATXN7-92Q NT with colloidal staining shows very similar ratios of the DUBm subunits. Lane M, molecular mass markers. (B) Immunoblot analysis (low and high exposures are shown) of the DUBm after double-affinity purification (A) verifies the identities and the presence of similar amounts of DUBm components. The different subunits were detected with anti-HA and anti-Flag antibodies, as indicated. *, modified forms of ATXN7-24Q NT or ATXN7-92Q NT due to ubiquitination (data not shown). (C) Comparative Ub-AMC hydrolysis assays using the reconstituted DUBm complexes with either ATXN7-24Q NT or ATXN7-92Q NT after the double-affinity purification confirm the very similar deubiquitinating activities between the two complexes. (D and E) Purification of reconstituted DUBm containing ATXN7-24Q NT (C) or ATXN7-92Q NT (D) by size exclusion chromatography using Superdex 200 gel filtration. Column fractions (numbers above the blots) were analyzed by immunoblotting with anti-HA (USP22) and anti-Flag (ATXN7, ATXN7L3, and ENY2) antibodies, and band positions for these components are as indicated. The elution profile of protein markers is indicated at the top of the blots. A complex containing all four subunits, including either ATXN7-24Q NT or ATXN7-92Q NT, is present in fractions 23 and 24 of both purifications and corresponds to the expected molecular mass for the DUBm. A darker exposure of the input lane for the ATXN7-92Q NT purification (labeled high) is provided to confirm the presence of all components in the starting material. ‡, degradation fragments. (F) Comparison of the integrity and stoichiometry of fraction 24 of the gel filtration from panels D and E was performed by immunoblotting with anti-HA and anti-Flag antibodies. Note that the amounts of the different subunits between the two reconstituted DUBm complexes are very similar. *, modified form of ATXN7-24Q NT or ATXN7-92Q NT due to ubiquitination (data not shown). (G and H) In vitro DUB assays using purified mononucleosomes as the substrate were performed with both DUB modules, and H2A and H2B ubiquitination levels were monitored with antibodies specific for these modifications. Both reconstituted complexes showed very similar enzymatic activities toward both ubiquitinated histone H2A and H2B incorporated into nucleosomes at the time points analyzed. H2A or H2B immunoblotting or Ponceau S staining served as a loading control.

FIG 4

Overexpression of ATXN7L3 and ENY2 prevents sequestration of ATXN7L3 into aggregates with ATXN7-92Q in vivo. (A) Exogenous expression of ATXN7-92Q, but not that of ATXN7-24Q, causes aggregate formation in human astrocytes. ATXN7L3 colocalization with nuclear inclusions in ATXN7-92Q-expressing astrocytes was observed by immunofluorescence staining using anti-Flag and anti-ATXN7L3 antibodies. Cooverexpression of ATXN7L3 and ENY2 reduces this colocalization by inhibiting ATXN7-92Q aggregation. Nuclei were stained with DAPI (blue). Bar, 10 μm. (B) Upon cooverexpression of ATXN7L3 and ENY2 with ATXN7-92Q, the amount of soluble ATXN7-92Q in the supernatant fraction increased, whereas the amount of SDS-insoluble ATXN7-92Q aggregates in the pellet (trapped in the stacking gel) dramatically decreased compared to the amount obtained with overexpression of ATXN7-92Q alone. ATXN7-24Q is highly soluble. Anti-β-actin immunoblotting served as a loading control. ATXN7-24Q and ATXN7-92Q were tagged with Flag. ATXN7L3 and ENY2 were tagged with a V5 epitope. (C) Quantification of astrocytes containing nuclear inclusions. One hundred cells from multiple fields were counted and then sorted by the number of nuclear inclusions per cell (N). Three independent experiments were performed, and a two-tailed Student t test was used for statistical analysis; error bars represent ±SDs. (D) Quantification of astrocytes with endogenous ATXN7L3 colocalization with nuclear inclusions. Three independent experiments were performed, and a two-tailed Student t test was used for statistical analysis; error bars represent SDs.

FIG 5

Solubility of ATXN7-92Q in vivo correlates with DUB activity. (A) Core histones were purified from human astrocytes infected with empty vector (lanes 1 to 3), ATXN7-24Q (lanes 4 to 6), or ATXN7-92Q (lanes 7 to 9) and were analyzed by electrophoresis of increasing sample amounts (1× to 3×), and the levels of H2Bub were monitored with antibodies specific for this modification. The levels of H2B ubiquitination increased in astrocytes expressing ATXN7-92Q, indicating impaired deubiquitination. Quantification of H2Bub levels was normalized to H2B levels using ImageJ software. (B) Cooverexpression of ATXN7L3 and ENY2 enhances global H2Bub deubiquitination in astrocytes expressing ATXN7-92Q, whereas H3K9/14ac levels are not altered. Different sample amounts (1× and 3×) were analyzed. H2B immunostaining or Ponceau S staining served as a loading control. (C) Core histones were purified from the cerebellums of 4-month-old mice bearing wild-type (5Q) or mutant (100Q) alleles of the ATXN7 gene. Both ATXN7^100Q/100Q and ATXN7^100Q/5Q mice exhibit SCA7 symptoms over time, but disease progression in ATXN7^100Q/100Q mice was faster and more severe than that in ATXN7^100Q/5Q mice. Immunoblot analysis was used to detect H2Bub levels with anti-H2Bub antibody. Samples from both ATXN7^100Q/100Q and ATXN7^100Q/5Q mice show increased H2B ubiquitination, with the effect being more severe in the samples from ATXN7^100Q/100Q mice. H2Bub levels were normalized to H2B levels using ImageJ software. Representative results chosen from more than three independent experiments are shown. (D) Model of the contribution of ATXN7-poly(Q) to SCA7 disease in humans. In vitro, soluble DUBm with ATXN7-92Q exhibits DUB activity comparable to that of the DUBm with ATXN7-24Q. However, in vivo ATXN7-poly(Q) tends to form aggregates that sequester DUBm components away from their substrates. Incorporation into DUBm can help solubilize ATXN7-poly(Q) in vitro and in vivo, partially alleviating the formation of the poly(Q)-containing aggregates in astrocytes. Impaired DUB activity in the cerebellums of SCA7 patients expressing ATXN7-poly(Q) likely leads to increased global H2Bub levels, as well as the deregulation of other DUBm substrates, contributing to SCA7 disease.