Autophagy can either be cytoprotective or promote cell death in a context-dependent manner in response to stress. How autophagy leads to autophagy dependent cell death requires further clarification. In this study, we document a nonlinear roller coaster form of autophagy oscillation when cells are subjected to different stress conditions. Serum starvation induces an initial primary autophagic peak at 6 h, that helps to replenish cells with de novo fluxed nutrients, but protracted stress lead to a secondary autophagic peak around 48 h. Time kinetic studies indicate that the primary autophagic peak is reversible, whereas the secondary autophagic peak is irreversible and leads to cell death. Key players involved in different stages of autophagy including initiation, elongation and degradation during this oscillatory sequence were identified. A similar molecular pattern was intensified under apoptosis-deficient conditions. mTOR was the central molecule regulating this autophagic activity, and upon knockdown a steady increase of autophagy without any non-linear fluctuation was evident. An unbiased proteome screening approach was employed to identify the autophagy molecules potentially regulating these autophagic peaks. Our proteomics analysis has identified Annexin A2 as a stress-induced protein to implicate in autophagy fluctuation and its deficiency reduced autophagy. Moreover, we report that mTOR in its phosphorylated condition interacts with Annexin A2 to induce autophagy fluctuation by altering its cellular localization. The work highlights the molecular mechanism of a mTOR-dependent roller coaster fluctuation of autophagy and autophagy dependent cell death during prolong stress.

自噬可以是细胞保护性的，也可以在依赖压力的情况下以依赖于上下文的方式促进细胞死亡。自噬如何导致自噬依赖性细胞死亡需要进一步阐明。在这项研究中，我们记录了当细胞经受不同的应力条件时，自噬振荡的非线性过山车形式。血清饥饿会在6 h引起一个初始的自噬峰，这有助于从头开始补充营养素，但长时间的应激会在48 h左右导致继发的自噬峰。时间动力学研究表明，一级自噬峰是可逆的，而二级自噬峰是不可逆的，并导致细胞死亡。确定了参与自噬不同阶段的关键参与者，包括在该振荡序列中的起始，延伸和降解。在凋亡不足的条件下，类似的分子模式被增强。mTOR是调节这种自噬活性的中心分子，并且在敲低后，自噬的稳定增加没有明显的非线性波动。采用无偏蛋白质组筛选方法来鉴定可能调节这些自噬峰的自噬分子。我们的蛋白质组学分析已将膜联蛋白A2鉴定为应激诱导的蛋白，提示其参与自噬波动，而其缺陷降低了自噬。此外，我们报告说mTOR在其磷酸化条件下与膜联蛋白A2相互作用，通过改变其细胞定位来诱导自噬波动。这项工作突出了长期应激期间自噬和自噬依赖性细胞死亡的mTOR依赖性过山车波动的分子机制。