title: Mass spectrometric identification of novel posttranslational modification sites in Huntingtin.
abstract: Huntington's disease (HD) is caused by a CAG triplet repeat expansion in exon 1 of the Huntingtin (Htt) gene, encoding an abnormal expanded polyglutamine (polyQ) tract that confers toxicity to the mutant Htt (mHtt) protein. Recent data suggest that posttranslational modifications of mHtt modulate its cytotoxicity. To further understand the cytotoxic mechanisms of mHtt, we have generated HEK293 cell models stably expressing Strep- and FLAG-tagged Htt containing either 19Q (wild-type Htt), 55Q (mHtt), or 94Q (mHtt) repeats. Following tandem affinity purification, the tagged Htt and associated proteins were subjected to tandem mass spectrometry or 2D nano-LC tandem mass spectrometry and several novel modification sites of mHtt containing 55Q or 94Q were identified. These were phosphorylation sites located at Ser431 and Ser432, and ubiquitination site located at Lys444. The two phosphorylation sites were confirmed by Western blot analysis using phosphorylation site-specific antibodies. In addition, prevention of phosphorylation at the two serine sites altered mHtt toxicity and accumulation. These modifications of mHtt may provide novel therapeutic targets for effective treatment of the disorder.
authors.affiliation: Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, SD 57069, USA.
authors.forename: Gaofeng
authors.initials: G
authors.lastname: Dong
authors.affiliation: Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, SD 57069, USA.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Callegari
authors.affiliation: Helmholtz Zentrum München, German Research Center for Environmental Health, Research Unit for Protein Science, Neuherberg, Germany.|Institute for Ophthalmic Research, Medical Proteome Center, University of Tubingen, Tubingen, Germany.
authors.forename: Christian J
authors.initials: CJ
authors.lastname: Gloeckner
authors.affiliation: Helmholtz Zentrum München, German Research Center for Environmental Health, Research Unit for Protein Science, Neuherberg, Germany.|Institute for Ophthalmic Research, Medical Proteome Center, University of Tubingen, Tubingen, Germany.
authors.forename: Marius
authors.initials: M
authors.lastname: Ueffing
authors.affiliation: Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, SD 57069, USA.
authors.forename: Hongmin
authors.initials: H
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D057809:HEK293 Cells; D006801:Humans; D000071058:Huntingtin Protein; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D053719:Tandem Mass Spectrometry / Q000379:methods*; D054875:Ubiquitination
chemical_list: C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins