Department of Neurobiology, Tel-Aviv University, Ramat-Aviv 69978, Israel.
J Cell Biochem. 2010 Aug 1;110(5):1262-71. doi: 10.1002/jcb.22642.
Autophagy, a process of self-digestion of cellular constituents, regulates the balance between protein synthesis and protein degradation. Beclin 1 represents an important component of the autophagic machinery. It interacts with proteins that positively regulate autophagy, such as Vps34, UVRAG, and Ambra1, as well as with anti-apoptotic proteins such as Bcl-2 via its BH3-like domain to negatively regulate autophagy. Thus, Beclin 1 interactions with several proteins may regulate autophagy. To identify novel Beclin 1 interacting proteins, we utilized a GST-Beclin 1 fusion protein. Using mass spectroscopic analysis, we identified Beclin 1 as a protein that interacts with GST-Beclin 1. Further examination by cross linking and co-immunoprecipitation experiments confirmed that Beclin 1 self-interacts and that the coiled coil and the N-terminal region of Beclin 1 contribute to its oligomerization. Importantly, overexpression of vps34, UVRAG, or Bcl-x(L), had no effect on Beclin 1 self-interaction. Moreover, this self-interaction was independent of autophagy induction by amino acid deprivation or rapamycin treatment. These results suggest that full-length Beclin 1 is a stable oligomer under various conditions. Such an oligomer may provide a platform for further protein-protein interactions.
自噬是细胞成分自我消化的过程，调节着蛋白质合成与降解之间的平衡。Beclin 1 是自噬机制中的一个重要组成部分。它通过 BH3 样结构域与抗凋亡蛋白（如 Bcl-2）相互作用，负向调节自噬，同时与正向调控自噬的蛋白（如 Vps34、UVRAG 和 Ambra1）相互作用。因此，Beclin 1 与多种蛋白的相互作用可能调节自噬。为了鉴定新的 Beclin 1 相互作用蛋白，我们利用 GST-Beclin 1 融合蛋白进行研究。通过质谱分析，我们鉴定出 Beclin 1 是与 GST-Beclin 1 相互作用的蛋白。交联和共免疫沉淀实验进一步证实了 Beclin 1 的自身相互作用，并且卷曲螺旋和 Beclin 1 的 N 端区域有助于其寡聚化。重要的是，vps34、UVRAG 或 Bcl-x(L) 的过表达对 Beclin 1 的自身相互作用没有影响。此外，这种自身相互作用不依赖于氨基酸剥夺或雷帕霉素处理诱导的自噬。这些结果表明，全长 Beclin 1 在各种条件下都是一种稳定的寡聚体。这种寡聚体可能为进一步的蛋白-蛋白相互作用提供了一个平台。
