G38R和D40G突变通过降低膜联蛋白A11的蛋白质稳定性导致肌萎缩侧索硬化症
[Mutations of G38R and D40G cause amyotrophic lateral sclerosis by reducing Annexin A11 protein stability].
作者信息
Liao Di, Liao Qiao, Huang Cao, Bi Fangfang
机构信息
Department of Neurology, Xiangya Hospital, Central South University, Changsha 410008, China.
Department of Pathology, Anatomy & Cell Biochemistry, Thomas Jefferson University, Philadelphia PA 19107, USA.
出版信息
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2018 Jun 28;43(6):577-582. doi: 10.11817/j.issn.1672-7347.2018.06.001.
To explore the role of the mutations G38R and D40G of Annexin A11 (ANXA11) in the onset of amyotrophic lateral sclerosis (ALS). Methods: The plasmids expressing ANXA11 wild type protein, ANXA11 G38R protein and ANXA11 D40G protein were constructed, respectively. The recombinant plasmids were then transfected into HEK293 cells respectively followed by cycloheximide (CHX) treatment for 0, 2, 4 and 8 h. The protein expressions of ANXA11 wild type, ANXA11 G38R and ANXA11 D40G mutations were determined by Western blot. Gray analysis by Image J was performed to compare the half-life of each protein. The NSC-34 cell lines constantly expressing ANXA11 wild type protein, ANXA11 G38R protein and ANXA11 D40G protein were established. The cells were treated with NP-40 lysis buffer to examine the protein solubility by Western blot. Results: Both ANXA11 G38R protein and ANXA11 D40G protein showed a shorter half-life than ANXA11 wild type protein (P<0.05), while there was no difference between ANXA11 G38R protein and ANXA11 D40G protein (P>0.05). There was no visible insoluble substance in the NP-40 lysates for ANXA11 wild type protein, ANXA11 G38R protein and ANXA11 D40G protein. Conclusion: G38R and D40G mutations reduce the stability of ANXA11 protein. G38R and D40G mutations do not alter ANXA11 solubility.
探讨膜联蛋白A11（ANXA11）的G38R和D40G突变在肌萎缩侧索硬化症（ALS）发病中的作用。方法：分别构建表达ANXA11野生型蛋白、ANXA11 G38R蛋白和ANXA11 D40G蛋白的质粒。然后将重组质粒分别转染至HEK293细胞，随后用环己酰亚胺（CHX）处理0、2、4和8小时。通过蛋白质免疫印迹法测定ANXA11野生型、ANXA11 G38R和ANXA11 D40G突变体的蛋白表达。使用Image J进行灰度分析以比较每种蛋白的半衰期。建立持续表达ANXA11野生型蛋白、ANXA11 G38R蛋白和ANXA11 D40G蛋白的NSC-34细胞系。用NP-40裂解缓冲液处理细胞，通过蛋白质免疫印迹法检测蛋白溶解度。结果：ANXA11 G38R蛋白和ANXA11 D40G蛋白的半衰期均短于ANXA11野生型蛋白（P<0.05），而ANXA11 G38R蛋白和ANXA11 D40G蛋白之间无差异（P>0.05）。ANXA11野生型蛋白、ANXA11 G38R蛋白和ANXA11 D40G蛋白的NP-40裂解物中均未观察到可见的不溶物。结论：G38R和D40G突变降低了ANXA11蛋白的稳定性。G38R和D40G突变不会改变ANXA11的溶解度。
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