One-Pot Semisynthesis of Exon 1 of the Huntingtin Protein: New Tools for Elucidating the Role of Posttranslational Modifications in the Pathogenesis of Huntington's Disease
One-Pot Semisynthesis of Exon 1 of the Huntingtin Protein: New Tools for Elucidating the Role of Posttranslational Modifications in the Pathogenesis of Huntington's Disease
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DOI：
10.1002/anie.201307510
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发表时间：
2014-02-10
影响因子：
16.6
通讯作者：
Lashuel, Hilal A.
中科院分区：
文献类型：
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作者：
Ansaloni, Annalisa;Wang, Zhe-Ming;Lashuel, Hilal A.
参与调节亨廷顿蛋白外显子1（Httex 1）前17个残基（Nt 17）内许多翻译后修饰（PTM）的天然酶仍然未知。开发了一种半合成策略，该策略允许通过使用表达蛋白连接（EPL）在Nt 17内位点特异性引入PTM。该策略用于产生含有23个谷氨酰胺残基的未加标签的野生型（wt）和T3-磷酸化（pT 3）Httex 1（Httex 1 - 23 Q）。我们的研究表明，pT 3显着减缓的寡聚化和纤维化的Httex 1 - 23 Q和Httex 1变体含有polyQ重复低于致病阈值容易聚集和形成原纤维体外。这些发现表明，跨越polyQ致病阈值对于Httex 1聚集不是必需的。产生WT或位点特异性修饰的无标签Httex 1的能力应有助于确定其结构和N-末端PTM在调节Htt在健康和疾病中的功能中的作用。
The natural enzymes involved in regulating many of the posttranslational modifications (PTMs) within the first 17 residues (Nt17) of Huntingtin exon1 (Httex1) remain unknown. A semisynthetic strategy that allows the site-specific introduction of PTMs within Nt17 by using expressed protein ligation (EPL) was developed. This strategy was used to produce untagged wild-type (wt) and T3-phosphorylated (pT3) Httex1 containing 23 glutamine residues (Httex1-23Q). Our studies show that pT3 significantly slows the oligomerization and fibrillization of Httex1-23Q and that Httex1 variants containing polyQ repeats below the pathogenic threshold readily aggregate and form fibrils invitro. These findings suggest that crossing the polyQ pathogenic threshold is not essential for Httex1 aggregation. The ability to produce wt or site-specifically modified tag-free Httex1 should facilitate determining its structure and the role of N-terminal PTMs in regulating the functions of Htt in health and disease.