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. 2017 Nov 1;7(1):14797.
doi: 10.1038/s41598-017-13711-7.

HSP90 recognizes the N-terminus of huntingtin involved in regulation of huntingtin aggregation by USP19

Wen-Tian He  1 , Wei Xue  1 , Yong-Guang Gao  1 , Jun-Ye Hong  1 , Hong-Wei Yue  1 , Lei-Lei Jiang  1 , Hong-Yu Hu  2
Affiliations

HSP90 recognizes the N-terminus of huntingtin involved in regulation of huntingtin aggregation by USP19

Wen-Tian He et al. Sci Rep. .

Abstract

Huntington's disease (HD) is caused by aberrant expansion of polyglutamine (polyQ) in the N-terminus of huntingtin (Htt). Our previous study has demonstrated that HSP90 is involved in the triage decision of Htt, but how HSP90 recognizes and regulates Htt remains elusive. We investigated the interaction between HSP90 and the N-terminal fragments of Htt (Htt-N), such as the N-terminal 90-residue fragment (Htt-N90). Our results showed that HSP90 binds to the N-terminal extreme of Htt-N in a sequence just ahead of the polyQ tract. Structural integration of the middle and C-terminal domains of HSP90 is essential for interacting with Htt-N90, and the dimerization mediated by the C-terminal domain facilitates this interaction. Moreover, ubiquitin-specific protease 19 (USP19), a deubiquitinating enzyme interacting with HSP90, up-regulates the protein level of Htt-N90 and consequently promotes its aggregation, whereas disruption of the interaction between Htt-N90 and HSP90 attenuates the effect of USP19 on Htt-N90. Thus, HSP90 interacts with Htt-N90 on the N-terminal amphipathic α-helix, and then recruits USP19 to modulate the protein level and aggregation of Htt-N90. This study provides mechanistic insights into the recognition between HSP90 and the N-terminus of Htt, and the triage decision for the Htt protein by the HSP90 chaperone system.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Supernatant/pellet fractionation showing that polyQ-expanded Htt sequesters chaperones into insoluble aggregates. (a) Domain architecture of the N-terminal fragment of Htt. The N-terminal 17-residue region is shown in detail. Qn, polyQ tract; PRR, proline-rich region. The normal 23 glutamines are included for numbering. (b) Sequestration of endogenous HSP90 and HSP70 by Htt-N552100Q. FLAG-tagged Htt-N55218Q or Htt-N552100Q was transfected into HEK 293 T cells, and the cell lysates were subjected to supernatant/pellet fractionation and Western blotting with anti-FLAG, anti-HSP90 and anti-HSP70 antibodies. (c,d) Quantification of the amounts of endogenous HSP90 (c) or HSP70 (d) in supernatant and pellet fractions. Data were from (b) and presented as Mean ± SEM (n = 3). **p < 0.01; ***p < 0.001; N.S., no significance. Vec., vector; Sup., supernatant; Pel., pellet.
Figure 2
Figure 2
The N-terminal fragments of Htt interact with HSP90. (a) GST pull-down experiment for the interactions of Htt-N9018Q or Htt-N17118Q with HSP90 in the presence of ATP or ADP. Bottom panel, SDS-PAGE with Coomassie blue staining; top panel, Western blotting to detect HSP90 with an anti-HSP90 antibody (5% input). (b) Examination of the interaction of Htt-N9018Q or its F11A/L14A mutant (Htt-N9018QM) with HSP90. HSP90 was detected by Western blotting with the anti-HSP90 antibody (top panel). M, mutant.
Figure 3
Figure 3
NMR titration showing the specific interaction between Htt-N20 and HSP90. (a) Plot of the relative peak intensities of amides against the residue number of C-terminal GB1-fused Htt-N20 upon titration with HSP90(1–696). The peak intensities were normalized as 1 for all peaks of free Htt-N20-GB1 except those of unassigned residues. (b) As in (a), the F11A/L14A mutant (Htt-N20M-GB1) upon titration with HSP90(1–696).
Figure 4
Figure 4
CD spectra exhibiting formation of an amphipathic α-helix in the N-terminal extreme. (a) Far-UV CD spectra of Htt-N20 with various concentrations. A cuvette with 1-mm path-length was used for recording at different concentrations, but for a concentration of 1.0 mg/mL, the CD signals at wavelengths below 210 nm were not recorded due to noise. (b) CD spectra of Htt-N20 in different concentration of TFE (v/v). The concentration of Htt-N20 peptide was 0.2 mg/mL. deg, degree. (c) The helical wheel plot for residues 4–17 of Htt-N20. The hydrophobic residues are shown in yellow, serines in green, glutamates in red, and lysines in blue.
Figure 5
Figure 5
Solution structure of Htt-N20 and NMR titration for the helical mutants. (a) Superposition of the backbone traces of the 10 lowest-energy structures. (b) Ribbon diagram of a representative structure of the Htt-N20 peptide. The structure was solved in a C-terminal GB1-fused peptide (Htt-N20-GB1). (c) Plot of the relative peak intensities of amides against the residue number of Htt-N20S13P-GB1 upon titration with HSP90(1–696). The average of the intensities for residues 4–17 was ~0.86 at a 1:1 ratio titration. (d) As in (c), Htt-N20A10P-GB1. (e) As in (c), Htt-N20A10P/S13P-GB1.
Figure 6
Figure 6
NMR titration suggesting that HSP90-MC is involved in the interaction with Htt-N20. (a,b) Plot of the relative peak intensities of amides against the residue number of Htt-N20-GB1 upon titration with HSP90-NM (residues 1–548) (a) or HSP90-MC (236–696) (b). (c) Elution profiles of HSP90-MC and its mutant (I688A/Y689A/I692A, HSP90-MCmut) by using size exclusion chromatography. Purified WT or mutant HSP90-MC protein (~100 μM) was loaded on a Superdex-200 Increase 10/300 GL column in a phosphate buffer (20 mM phosphate, 50 mM NaCl, pH 6.5). A280, Absorbance at 280 nm; mAU, milli absorbance unit. (d) As in (a), Htt-N20-GB1 upon titration with HSP90-MCmut.
Figure 7
Figure 7
Interaction with HSP90 is essential for regulation of the protein level of Htt-N9018Q by USP19_b. (a) Effect of USP19_b on the protein level of Htt-N9018Q in a dose-dependent manner. Htt-N9018Q-GFP was co-transfected with different dose of HA-USP19_b into HEK 293 T cells. The amounts of Htt-N9018Q were detected by Western blotting with an anti-GFP antibody. (b) As in (a), effect of USP19_b on the protein level of the F11A/L14A mutant (Htt-N9018QM-GFP). (c) As in (a), effect of USP19_b on the protein level of the N-terminally 14-residue deleted mutant (Htt-N9018QΔN-GFP). (d) Quantification of the amounts of Htt-N9018Q-GFP and its mutants affected by USP19_b. Data were from (a), (b) and (c), and presented as Mean ± SEM (n = 3). ***p < 0.001.
Figure 8
Figure 8
The promoting effect of USP19_b on the aggregation of Htt-N90100Q is dependent on the HSP90-Htt-N interaction. (a) Effect of USP19_b on the aggregation of Htt-N90100Q and its mutants. FLAG-tagged Htt-N90100Q (WT), its F11A/L14A mutant (M) or N-terminally 14-residue deleted mutant (ΔN) was co-transfected with HA-USP19_b into HEK 293 T cells. The cell lysates were subjected to filter trap and Western blotting with an anti-FLAG antibody. (b) Quantification of the aggregates of Htt-N90100Q and its mutants affected by USP19_b. Data were from (a) and presented as Mean ± SEM (n = 3). **p < 0.01; N.S., no significance. Vec., vector. (c) Schematic representation for recognition and triage decision of Htt modulated by USP19 through the HSP90 chaperone. HSP90 (blue) recognizes the client protein Htt (brown) and then recruits the deubiquitinating enzyme USP19 through the CS domains (orange) to regulate the ubiquitination state of Htt. These processes ultimately determine the triage of Htt for stabilization, aggregation or degradation.
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