Polyglutamine Tract Expansion Increases S-Nitrosylation of Huntingtin and Ataxin-1

<div><p>Expansion of the polyglutamine (polyQ) tract in the huntingtin (Htt) protein causes Huntington’s disease (HD), a fatal inherited movement disorder linked to neurodegeneration in the striatum and cortex. S-nitrosylation and S-acylation of cysteine residues regulate many functions of cytosolic proteins. We therefore used a resin-assisted capture approach to identify these modifications in Htt. In contrast to many proteins that have only a single S-nitrosylation or S-acylation site, we identified sites along much of the length of Htt. Moreover, analysis of cells expressing full-length Htt or a large N-terminal fragment of Htt shows that polyQ expansion strongly increases Htt S-nitrosylation. This effect appears to be general since it is also observed in Ataxin-1, which causes spinocerebellar ataxia type 1 (SCA1) when its polyQ tract is expanded. Overexpression of nitric oxide synthase increases the S-nitrosylation of normal Htt and the frequency of conspicuous juxtanuclear inclusions of Htt N-terminal fragments in transfected cells. Taken together with the evidence that S-nitrosylation of Htt is widespread and parallels polyQ expansion, these subcellular changes show that S-nitrosylation affects the biology of this protein <i>in vivo</i>.</p></div

Full-length Htt and Htt fragments.

<p>(A) Wild-type (wt) full-length Htt. The vertical lines indicates all 70 Cys residues. The horizontal open boxes indicate HEAT repeat motif clusters (Tartari, M. <i>et al</i>., <i>Mol Biol Evol</i>. 2008). The horizontal grey bar labels caspase cleavage sites. Anti-Htt antibodies (MAB2166, MCA2050, and MAB2168) and their binding sites are labeled. (B) Htt N548 fragment (1–548 a.a.) and its posttranslational modifications. The inset is an enlargement of this region. Cysteine residue numbers are indicated. (C) Full-length (FL) Htt constructs for transient expression. (D) Htt N-terminal 1–548 a.a. fragments that approximate proteolytic products of full-length Htt. (E) Constructs for expressing Htt exon1 coding region (1–90 a.a.). (F) C-terminal construct (585–3144 a.a.).</p

The C214S mutation reduces Htt S-nitrosylation and S-acylation in the context of the normal polyQ tract.

<p>(A) S-nitrosylation (SNO) and S-acylation (S-acyl) of Htt N548Q15, N548Q15-C214S, N548Q128, and N548Q128-C214S. Recombinant proteins were expressed in COS7 cells for one day. The stars indicate significant reduction due to the C214S mutation. C: C214; S: C214S. (B) Quantification of the ratio of Htt N548 S-nitrosylation to total N548 (n = 3). The inset enlarges the scale for N548Q15 and N548Q15-C214S. (C) Quantification of the ratio of Htt N548 S-acylation to the total N548 (n = 3). In (B) and (C), experiments for C214 mutants were performed in parallel with Cys wild-type shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163359#pone.0163359.g002" target="_blank">Fig 2A</a>. <i>p</i>-values from t-test are indicated if <i>p</i><0.05. The error bar represents the SEM.</p

Summary of SNO and S-acylation sites in Htt N548Q15 and N548Q128.

<p>Summary of SNO and S-acylation sites in Htt N548Q15 and N548Q128.</p

PolyQ expansion increases S-nitrosylation of Htt and Ataxin-1.

<p>Recombinant proteins were expressed in cells for one day. SNO-RAC and acyl-RAC were used to recover S-nitrosylated and S-acylated proteins, respectively. The negative control shows that non-specific binding is negligible. Western blotting was used to detect Htt or Ataxin-1 (FLAG-tagged). (A) SNO and S-acylation of Htt N548 fragments expressed in COS7 cells. The longer exposure of the film shows the weak SNO signal from N548 with a normal polyQ tract. (B) Quantification of S-nitrosylated and S-acylated N548 expressed in COS7 cells (n = 3). (C) SNO and S-acylation of Htt N548 fragments expressed in HEK293T cells. (D) Quantitation of S-nitrosylated and S-acylated N548 expressed in HEK293T cells (n = 6 for S-nitrosylation and n = 5 for S-acylation). (E) SNO and S-acylation of Ataxin-1 proteins expressed in HEK293T cells. (F) Quantitation of S-nitrosylated and S-acylated Ataxin-1 expressed in HEK293T cells (n = 3). SNO: S-nitrosylation. S-acyl: S-acylation. ImageJ was used to determine band intensity in all figures. <i>p</i>-values from t-test are indicated if p<0.05. Error bars represent SEM in all figures.</p

PolyQ expansion in Ataxin-1 and Htt N548 proteins increases S-nitrosylated and S-acylated high molecular weight (HMW) species.

<p>SNO-RAC and acyl-RAC were used to recover S-nitrosylated and S-acylated proteins followed by Western blotting. (A) Top: S-nitrosylated and S-acylated monomer and high molecular weight (HMW) species of Ataxin-1 expressed in HEK293T cells. Bottom: HMW/monomer quantity ratios for Ataxin-1Q85 (n = 5). All experiments show that HMW material is enriched in S-nitrosylated and S-acylated proteins. (B) S-nitrosylated and S-acylated monomer and HMW species of Htt N548 expressed in HEK293T cells.</p