title: Polyglutamine expansion of ataxin-3 alters its degree of ubiquitination and phosphorylation at specific sites.
abstract: Ubiquitination and phosphorylation of proteins represent post translational modifications (PTMs) capable of regulating a variety of cellular processes. In the neurodegenerative disorder spinocerebellar ataxia type 3 (SCA3), the disease causing protein ataxin-3 carries an expanded polyglutamine (polyQ) stretch causing it to aggregate in nuclear inclusions. These inclusions are decorated with ubiquitin suggestive of a malfunction in the clearance of the mutant protein. Differences in ubiquitin chain topology between normal and polyQ expanded ataxin-3 could be involved in the differential clearance of the two proteins and play a role in SCA3 pathogenesis. Likewise, changes in phosphorylation patterns between the two variants could contribute to pathogenic processes involved in SCA3. We therefore determined the ubiquitination and phosphorylation patterns, together with the ubiquitin-linkage types associated with normal and polyQ expanded ataxin-3 by mass spectrometry (MS). This analysis revealed a similar ubiquitin linkage pattern on normal and expanded ataxin-3. However, the distribution of ubiquitinated lysine residues was altered in polyQ expanded ataxin-3, with increased ubiquitination at the new identified ubiquitination site lysine-8. MS analysis of phosphorylation also revealed novel phosphorylation sites in ataxin-3, and an increase in phosphorylation of expanded ataxin-3 at several positions. The study suggests that differences in clearance of normal and expanded ataxin-3 are not attributed to differences in ubiquitin-linkage pattern. However, the observed differences between the normal and polyQ expanded ataxin-3 with respect to the degree of ubiquitination and phosphorylation on specific sites may have an impact on ataxin-3 function and SCA3 pathogenesis.
authors.affiliation: Department of Neurodegeneration, H. Lundbeck A/S, Ottiliavej 9, 2500 Valby, Denmark; Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen N, Denmark. Electronic address: linevkristensen@yahoo.dk.
authors.forename: Line V
authors.initials: LV
authors.lastname: Kristensen
authors.affiliation: Evotec München GmbH, Am Klopferspitz 19a, 82152 Martinsried, Germany.
authors.forename: Felix S
authors.initials: FS
authors.lastname: Oppermann
authors.affiliation: Department of Neurodegeneration, H. Lundbeck A/S, Ottiliavej 9, 2500 Valby, Denmark; Department of Cellular and Molecular Medicine, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N, Denmark.
authors.forename: Matthias J
authors.initials: MJ
authors.lastname: Rauen
authors.affiliation: Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen N, Denmark.
authors.forename: Rasmus
authors.initials: R
authors.lastname: Hartmann-Petersen
authors.affiliation: Department of Neurodegeneration, H. Lundbeck A/S, Ottiliavej 9, 2500 Valby, Denmark.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Thirstrup
mesh_terms: D000595:Amino Acid Sequence; D000067699:Ataxin-3 / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D010455:Peptides / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology*
chemical_list: D010455:Peptides; D012097:Repressor Proteins; C097188:polyglutamine; C092341:ATXN3 protein, human; D000067699:Ataxin-3
keywords: Ataxin-3; Mass spectrometry; Neurodegeneration; Phosphorylation; Spinocerebellar ataxia type 3; Ubiquitination