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.
Highlights
Tional disturbances, and psychological deterioration [1]
Novel Lysine Acetylation Sites in Huntingtin lished preferences are that mitochondrial substrates with histidine and tyrosine at the ϩ1 position and sites found on protein surfaces [35, 45]
Consistent with our hypothesis that histone deacetylases (HDACs) inhibition may act through modification of huntingtin protein (Htt), we found that the levels of Htt acetylation increased when HEK293 were cultured in the presence of HDAC inhibitors (Fig. 5B)
Summary
Site-directed Mutagenesis—Site-directed mutagenesis of the Htt constructs pTet-c-Myc-Htt23Q and pTet-c-Myc-Htt148Q [20] was performed using the QuikChange kit (Stratagene, La Jolla, CA) using the following primers: a double stop was inserted after amino acid 612, forward, 5Ј-CCACAGGTA-TTCTTCCTTAGTAAG-CCTCGGAGGCCTTCAGG-3Ј, reverse, 5Ј-CCTGAAGGCC-TCCGAGGCTTACTAA-GGAAGAATACCTGTGG-3Ј. The following search parameters were used: enzyme specificity was defined as trypsin with two possible missed cleavages, carbamidomethyl (Cys) was chosen as fixed modification, acetyl (Protein N-terminal), Gln-Ͼpyro-Glu (N-terminal Gln), and oxidation (Met) were chosen as variable modifications, mass tolerance for precursor ions was 50 ppm, mass tolerance for fragment ions was 0.4 Da. The publicly available SwissProt database release version 56.2 (release 23-Sep-2008) was searched for most data obtained from human samples with species restriction “human” (20407 sequences); Ions scores are [-10*Log(P)], where P is the probability that the observed match is a random event. The following search parameters were used: enzyme specificity was defined as trypsin, Asp-N, chymotrypsin with three possible missed cleavages (supplemental Table S5), respectively, carbamidomethyl (Cys) was chosen as fixed modification, acetyl (Lys), acetyl (Protein N-terminal), Gln-Ͼpyro-Glu (N-terminal Gln), and oxidation (Met) were chosen as variable modifications, mass tolerance for precursor ions was 0.4 Da, mass tolerance for fragment ions was 0.4 Da
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