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帕金森氏病相关蛋白Pink1促进自噬的研究
Autophagy Promoted by Pakinson’s Disease Related Protein Pink1
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摘要: 目的 探讨帕金森氏病相关蛋白Pink1在自噬过程的作用。 方法 采用RT-PCR扩增基因片段,构建Pink1﹑自噬抑制蛋白Bcl-XL和自噬激活因子Beclin1等重组质粒,并通过慢病毒系统建立稳定表达Pink1-Flag的HEK293细胞株,通过重组质粒转染细胞,过表达相关蛋白,用免疫共沉淀和Western blot检测Pink1和Bcl-XL的相互作用,Pink1对Bcl-XL和Beclin1相互作用的影响,以及Pink1对自噬的影响。 结果 自噬抑制蛋白Bcl-XL与Pink1有相互作用,过量表达Pink1可以抑制Bcl-XL与自噬激活因子Beclin1的相互作用。过量表达Pink1提高自噬下游蛋白LC3Ⅱ/LC3Ⅰ。 结论 帕金森氏病相关蛋白Pink1通过与自噬抑制蛋白Bcl-XL相互作用释放自噬促进因子Beclin1来激活自噬。Abstract: Objecitve To investigate the function of Parkinson's disease(PD)-related protein Pink1 in autophagy. Methods Pink1, autophagy inhibitor Bcl-XL and autophagy induced-factor Beclin1 were amplified with RT-PCR and contructed into pcDNA3.1(+)vector. The stable HEK293 cell line of Pink1 expression was established through the lentivirus system. Pink1, Bcl-XL and Beclin1 were tansfected into the HEK293 Cell Line. Co-Immunoprecipitation and Western blot were performed to determine the interaction between Pink1 and Bcl-XL, the effect of Pink1 on the interaction between Bcl-XL and Beclin1,and the function of Pink1 in autophagy. Results There was a new interaction between Pink1 and antophagy inhibitor Bcl-XL. Overexpressed Pink1 promoted the dissociation of autophagy induced-factor Beclin1 from Bcl-XL. Overexpressed Pink1 increased the autophagic protein LC3Ⅱ/LC3Ⅰ. Conclusion Pakinson's disease related protein Pink1 promotes the dissociation of autophagy induced-factor Beclin1 from antophagy inhibitor Bcl-XL and promotes autophagy.
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