Facile 鈥渟top codon鈥?method reveals elevated neuronal toxicity by discrete S87p-伪-synuclein oligomers

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• 作者：Yonghwang Ha ; Aerin Yang ; Seyoung Lee ; Kibong Kim ; Hyunjeong Liew ; Yoo-Hun Suh ; Hee-Sung Park ; David G. Churchill
• 关键词：Stop codon ; Phosphorylation ; Oligomerization ; Pyridoxal 5&prime ; -phosphate ; Copper ; Dopamine
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：17 January, 2014
• 年：2014
• 卷：443
• 期：3
• 页码：1085-1091
• 全文大小：1635 K

文摘

Herein, a new method for preparing phosphorylated proteins at specific sites has been applied to 伪-synuclein (伪-Syn). Three different 伪-Syn species phosphorylated at Serine 87 (S87p-伪-Syn), Serine 129 (S129p-伪-Syn) and Serine 87/129 (S87p,129p-伪-Syn) were prepared through the 鈥榮top codon鈥?method and verified by LC/MS/MS and immunoblotting. Each type of phosphorylated 伪-Syn was tested for oligomerization trends and cellular toxicity with dopamine (DA), Cu²⁺ ions and pyridoxal 5鈥?phosphate. Aggregation trends induced by DA or DA/Cu²⁺ were similar between phosphorylated and non-phosphorylated 伪-Syn in SDS-PAGE. However, except for the monomer, phosphorylated oligomers showed higher toxicity than the non-phosphorylated 伪-Syn (Np-伪-Syn) oligomers via WST-1 assays when tested on SH-SY5Y human neuroblastoma cells. In particular, S87p-伪-Syn and S87p,129p-伪-Syn oligomers induced by DA/Cu²⁺, showed higher toxicity than did S129p-伪-Syn. When 伪-Syn was treated with pyridoxal 5鈥?phosphate in the presence of DA or Cu²⁺ to determine aggregation effects, high inhibition effects were shown in both non-phosphorylated and phosphorylated versions. 伪-Syn co-incubated with DA or DA/Cu²⁺ showed less cellular toxicity upon pyridoxal 5鈥?phosphate treatment, especially in the case of DA-induced Np-伪-syn. This study supports that phosphorylated oligomers of 伪-Syn at residue 87 can contribute to neuronal toxicity and the pyridoxal 5鈥?phosphate can be used as an inhibitor for 伪-Syn aggregation.