Mass spectrometric identification of novel lysine acetylation sites in huntingtin

Abstract

Huntingtin (Htt) is a protein with a polyglutamine stretch in the N-terminus and expansion of the polyglutamine stretch causes Huntington's disease (HD). Htt is a multiple domain protein whose function has not been well characterized. Previous reports have shown, however, that post-translational modifications of Htt such as phosphorylation and acetylation modulate mutant Htt toxicity, localization, and vesicular trafficking. Lysine acetylation of Htt is of particular importance in HD as this modification regulates disease progression and toxicity. Treatment of mouse models with histone deacetylase inhibitors ameliorates HD-like symptoms and alterations in acetylation of Htt promotes clearance of the protein. Given the importance of acetylation in HD and other diseases, we focused on the systematic identification of lysine acetylation sites in Htt23Q (1-612) in a cell culture model using mass spectrometry. Myc-tagged Htt23Q (1-612) overexpressed in the HEK 293T cell line was immunoprecipitated, separated by SDS-PAGE, digested and subjected to high performance liquid chromatography tandem MS analysis. Five lysine acetylation sites were identified, including three novel sites Lys-178, Lys-236, Lys-345 and two previously described sites Lys-9 and Lys-444. Antibodies specific to three of the Htt acetylation sites were produced and confirmed the acetylation sites in Htt. A multiple reaction monitoring MS assay was developed to compare quantitatively the Lys-178 acetylation level between wild-type Htt23Q and mutant Htt148Q (1-612). This report represents the first comprehensive mapping of lysine acetylation sites in N-terminal region of Htt.

Fig. 1.

Immunoprecipitation and Western blot analysis of Htt. Myc-tagged Htt (1–612) constructs (23Q and 148Q) were expressed in 293T cells. A, Lysate input and flow-through (FT) during the immunoprecipitation procedure and (B) Immunoprecipitated Htt were probed with monoclonal N-terminal Htt antibody 2166. C, Immunoprecipitation and 1D SDS-PAGE of expressed Htt stained with Sypro Ruby. Myc-tagged Htt23Q (1–612) expressed in 293T cells was immunoprecipitated from cellular lysates using the Pierce Profound Mammalian C-myc tag IP/Co-IP kit. Samples were separated by one-dimensional SDS-PAGE on 4–12% Bis-Tris gels. Gels were stained with fluorescent SyproRuby protein gel stain (Invitrogen).

Fig. 2.

Three novel Htt acetylation sites identified by mass spectrometry. A, ESI-MS/MS tandem mass spectra of Lys-acetylated peptide DTSLKGSFGVTRK_AcEM (residues 333–347) obtained after Asp-N digestion of immunoprecipitated Htt23Q (1–612) with high (A) and low (B) collision energy. The molecular ion [M+3H]³⁺ at m/z 566.61 (M = 1696.80) was selected for CID. B, ESI-MS/MS tandem mass spectra of Lys-acetylated peptide EIKK_AcNGAPR (residue 175–183) obtained after trypsin digestion of immunoprecipitated Htt23Q (1–612). The molecular ion [M+3H]³⁺ at m/z 352.21 (M = 1053.60) was selected for CID. C, ESI-MS/MS tandem mass spectra of Lys-acetylated peptide AAAVPK_AcIM (residues 231–238) obtained after chymotrypsin digestion of immunoprecipitated Htt23Q (1–612). The molecular ion [M+2H]²⁺ at m/z 429.73 (M = 857.44) was selected for CID. K*: In all three spectra, is the diagnostic immonium for acetyllysine at m/z 126.1.

Fig. 3.

ESI-MS/MS tandem mass spectra of the acetylated lysine peptide EKLMK_AcAF (residue 5–11) obtained after Asp-N digestion of immunoprecipitated Htt23Q (1–612). A, The molecular ion [M+2H]²⁺ at m/z 454.74 (M = 907.47 Da) was selected for CID. K* refers to the two immonium ions for acetyllysine at m/z 126.1 and 143 under Q2 transmission setting 1 described in supplemental Table S2. B, The molecular ion [M+2H]²⁺ at m/z 454.74 (M = 907.47 Da) was selected for CID. K* refers to the two immonium ions for acetyllysine at m/z 126.1 and 143 under Q2 transmission setting 2 described in supplemental Table S2.

Fig. 4.

Western blot analysis of Htt with anti-acetyllysine antibodies. Htt15Q and Htt138Q (1–1212) were expressed in 293T cells and lysates were probed with anti-acetyllysine-236, 345, 444 polyclonal antibodies and N-terminal Htt antibody 2166.

Fig. 5.

Representative MRM extracted ion chromatograms (XIC) for wild type (Htt23Q) and mutant (Htt148Q) peptide. A, MRM extracted ion chromatograms (XIC) for wild type (Htt23Q) and mutant (Htt148Q) peptide ¹⁷⁵EIKK_AcNGAP¹⁸³R containing Lys-178 for each transition. Three transitions were selected for our analysis as depicted by the three panels. This was used in our quantitative analysis of the ratio of Htt23Q to Htt148Q. B, MRM extracted ion chromatograms (XIC) for wild-type (Htt23Q) and mutant (Htt148Q) peptide ¹⁷⁵EIKK_AcNGAP¹⁸³R containing Lys-178 for each transition in the presence of HDAC inhibitors.

Fig. 6.

Summary scheme of Htt PTMs. A, Schematic of Htt structure with four HEAT repeat domains (HH) and caspase/calpain domain. Red arrows indicate lysine acetylation sites identified by MS/MS in this study. B, N-terminal fragment of mutant Htt has enhanced toxicity.