Beclin 1的磷酸化对其介导的自吞噬及受辐照细胞命运的影响与机制研究

Autophagy is a ubiquitous highly conserved pathway in eukaryotic cells that takes place as a response to a variety of conditions, such as nutrient deprivation, growth factors withdrawal, energetic, and oxidative stress. Most recent research demonstrated that DNA damage can induce the autophagy initiation, but the molecular mechanism remained unclear. Our lab found recently that the autophagy inhibitor 3-MA which targeting Beclin 1/Vps34 has radiosensiting effect and serum starvation indued autophagy has radioprotective effect. Furthermore, we identified three new phosphorylation sites in Beclin 1 which were not reported: T57, T62, S64. Based on these results, we plan to conduct the following research: 1) Elucidating mechanism of radioprotective role of Beclin 1 and its phosphorylation; 2) Elucidating the role of ATM and DNA-Pkcs in radiation induced autophagy and proving if they phosphorylate Beclin 1 on T57, T62 or S64 after radiation. 3) Elucidating whether T57, T62 and S64 phosphorylation influence the dynamic interaction between Beclin 1, Vps34 and Bcl-2. After accomplishment of above research, we will not only demonstrate the role of Beclin 1 mediated autophagy in cell fate determination after radiation but also pave the way to develop new-concept radiosensitizing and radioprotective drugs.

自吞噬是一种真核细胞在营养缺失等条件下发生的一种保护性反应。最近证实，DNA损伤可以诱发细胞发生自吞噬，但其分子机制不太清楚。我们发现，采用针对Beclin 1/Vps34的自吞噬抑制剂3-MA可以起到辐射增敏作用，而诱导自吞噬则可以起到辐射保护作用。另外也发现了Beclin 1的3个新的磷酸化修饰位点T57、T62、S64。基于此，拟开展以下研究：1）阐明Beclin 1及其磷酸化修饰位点在电离辐射诱发细胞自吞噬中的作用和辐射防护意义；2）阐明ATM和DNA-PKcs在电离辐射作用下是否磷酸化Beclin 1及在诱发自吞噬中的作用；3）以Bcl-2及Vps34相互作用的动态调节为关键点，阐明磷酸化修饰对Beclin 1介导自吞噬的下游调节功能机制。通过该研究，不仅可以阐明Beclin 1介导的自吞噬在决定电离辐射之后细胞命运中的作用，还可以为研制新型辐射防护及辐射增敏药物提供理论基础。

自噬是一种真核细胞在营养缺失等条件下发生的保护型反应，研究表明DNA损伤可以诱导细胞发生自噬。我们前期研究发现，激活自噬可以起到辐射防护作用；而3-甲基腺嘌呤（3-MA）通过抑制Beclin 1-Vps34的自噬活性，表现出显著的辐射增敏作用。提示自噬在电离辐射之后细胞命运的决定中起着至关重要的作用。为了阐明Beclin 1介导的自噬在电离辐射之后细胞命运决定中的作用及分子机制，我们开展了以下研究： ①采用CRISPR-CAS9技术成功构建了Beclin1敲除的细胞系。②证实了Beclin1在肿瘤细胞增殖、迁移和侵袭中的重要作用。③阐明了Beclin1参与调控电离辐射引起的G2/M细胞周期阻滞。④证明了Beclin1调控电离辐射诱导自噬的重要机制。⑤探讨了Beclin1在电离辐射诱导的DNA损伤修复中的重要作用。本研究不但阐明了Beclin 1介导的自噬对细胞辐射敏感性影响的分子机制，还为筛选针对自噬的辐射增敏及辐射防护药物提供理论基础。