Kristensen Line V, Oppermann Felix S, Rauen Matthias J, Hartmann-Petersen Rasmus, Thirstrup Kenneth
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.
Evotec München GmbH, Am Klopferspitz 19a, 82152 Martinsried, Germany.
Neurochem Int. 2017 May;105:42-50. doi: 10.1016/j.neuint.2016.12.019. Epub 2017 Jan 6.
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.
蛋白质的泛素化和磷酸化代表了能够调节多种细胞过程的翻译后修饰（PTM）。在神经退行性疾病3型脊髓小脑共济失调（SCA3）中，致病蛋白ataxin-3携带一段扩展的聚谷氨酰胺（polyQ）序列，导致其在核内聚集体中聚集。这些聚集体被泛素修饰，提示突变蛋白清除存在功能障碍。正常和polyQ扩展的ataxin-3之间泛素链拓扑结构的差异可能参与了这两种蛋白的差异清除，并在SCA3发病机制中起作用。同样，这两种变体之间磷酸化模式的变化可能有助于SCA3相关的致病过程。因此，我们通过质谱（MS）确定了正常和polyQ扩展的ataxin-3的泛素化和磷酸化模式，以及与之相关的泛素连接类型。该分析揭示了正常和扩展的ataxin-3上类似的泛素连接模式。然而，polyQ扩展的ataxin-3中泛素化赖氨酸残基的分布发生了改变，在新鉴定的泛素化位点赖氨酸-8处泛素化增加。磷酸化的MS分析还揭示了ataxin-3中的新磷酸化位点，以及扩展的ataxin-3在几个位置的磷酸化增加。该研究表明，正常和扩展的ataxin-3清除差异并非归因于泛素连接模式的差异。然而，正常和polyQ扩展的ataxin-3在特定位点的泛素化和磷酸化程度上观察到的差异可能会影响ataxin-3的功能和SCA3发病机制。
