基础医学与临床 ›› 2007, Vol. 27 ›› Issue (6): 601-604.

• 研究论文 •    下一篇

a-synuclein磷酸化修饰提高多巴胺能神经细胞TH的活性

吴波(北京) 赵焕英 赵春礼 杨慧   

  1. 首都医科大学北京神经科学研究所 首都医科大学北京神经科学研究所 首都医科大学北京神经科学研究所 首都医科大学北京神经科学研究所
  • 收稿日期:2007-01-17 修回日期:2007-03-13 出版日期:2007-06-25 发布日期:2007-06-25
  • 通讯作者: 吴波(北京)

a-synuclein phosphorylation increased TH activity in MN9D cells

  

  • Received:2007-01-17 Revised:2007-03-13 Online:2007-06-25 Published:2007-06-25

摘要: 目的 观察突触核蛋白(α-synuclein, α-Syn)第129位丝氨酸(Ser129)磷酸化修饰对酪氨酸羟化酶(tyrosine hydroxylase, TH)活性的影响。方法 应用重叠延伸PCR定点突变法将129位丝氨酸编码碱基TCT突变为天冬氨酸(Asp,D)编码碱基GAT,获得编码模拟磷酸化α-Syn (S129D α-Syn)的DNA序列,插入逆转录病毒真核表达载体(pLNCX2)。包装逆转录病毒颗粒并感染多巴胺能神经细胞MN9D。通过实时定量RT-PCR鉴定α-Syn基因表达,Western blot检测TH磷酸化水平。结果 pLNCX2-α-Syn (wild type/S129D) 质粒测序结果正确,并在MN9D细胞中均过表达。野生型α-Syn过表达组同正常对照组相比TH磷酸化水平降低(p<0.01),而S129D α-Syn组TH的磷酸化水平同正常对照组相比明显升高(p<0.05)。结论 在MN9D细胞中,野生型α-Syn 抑制TH的活性,而α-Syn Ser129 磷酸化后TH活性明显升高。

Abstract: Objective To observe the effect of phosphorylated α-Syn on TH activity. Methods pLNCX2-S129D α-Syn vector was constructed using overlapping extension polymerase chain reaction (OE-PCR). The retroviral particles were packaged and infected MN9D cells. Expression of α-Syn in MN9D was tested using real time RT-PCR. Phosphorylated TH protein level was tested using Western blot. Results Sequences of wild-type and S129D α-Syn were both correct. The result of real time RT-PCR showed that wild-type and S129D α-Syn genes were overexpressed in MN9D compared with uninfected MN9D and vector control group. TH phosphorylation was reduced in wild-type α-Syn group, while increased significantly in S129D α-Syn group. Conclusion α-Syn phosphorylation involves in regulation of TH activity in MN9D cells.