能量匮乏条件下Mec1/ATR在细胞核、线粒体、自噬体和溶酶体/液泡互作过程中的功能及其分子机制的研究

The cells will start a precise and effective emergency pathway such as inducing autophagy, changing the interaction network of organelles to face and adapt to changes in the external environment. A large number of experimental studies have shown that if the cells are not given timely and correct response, the cells will not be able to exercise normal physiological functions and even die. In previous studies, we found that DNA damage sensor protein Mec1 is exported from the nucleus, subsequent specifically recruited to the mitochondria under energy deprivation condition. As starvation time proceeds, Mec1 leaves the mitochondria, some of which are degraded by autophagy, and some of them re-enter into the nucleus. Further studies have shown that the recruitment of Mec1 into mitochondria is important for maintaining mitochondrial morphology, respiration and autophagosome formation under energy deprivation condition. in addition, we also found that autophagy is closely related to the clearance of nuclear damaged DNA. It shows that Mec1 plays a very important role in the communication of nucleus, mitochondria, autophagy and vacuoles under energy deprivation condition. This project will be on the basis of previous research, and combined with the mammalian ATR stable knockdown cell line to elaborate the function, molecular mechanism and physiological function of Mec1/ATR in the interaction of nucleus, mitochondria, autophagosome and lysosome/vacuole during energy deprivation.

细胞在受到外界压力时会启动体内一套精准而又高效的应急装置如诱导细胞自噬，改变细胞器互作网络等方式来应对并适应外界环境变化。大量研究表明，细胞如果得不到及时正确的应答，将不能行使正常的生理功能甚至死亡。在前期的研究中，我们发现在能量匮乏条件下，DNA损伤感应蛋白Mec1从细胞核中出来并特异招募到线粒体上，随着饥饿时间的进行，Mec1离开线粒体，一部分被自噬所降解，一部分再次进入到细胞核中。进一步研究发现， Mec1出核招募到线粒体对于维持线粒体的形态、呼吸作用和自噬体形成非常重要。此外，我们还发现细胞自噬与受损DNA的清除有密切关系，表明Mec1在细胞核、线粒体、自噬体和液泡互作过程中扮演着非常重要的角色。本课题将在前期的研究基础上，并同时结合哺乳动物同源物ATR稳定敲低细胞株，全面阐述能量匮乏条件下Mec1/ATR在细胞核、线粒体、自噬体和溶酶体/液泡互作过程中的作用、分子机制及生理功能。

在本课题的研究中，我们发现了核运出因子Cse1参与了Mec1的出核，Mec1不出核，则能量匮乏诱导的自噬无法发生。除了以前发现的Ggc1负责招募Mec1招募到线粒体外，我们还发现线粒体蛋白Abf2参与了Mec1招募到线粒体上，并为能量匮乏诱导的自噬所必需，有意思的是，我们还发现Mec1招募到线粒体上受到ROS的调控，在加入ROS清除剂BHA后，能量匮乏诱导的自噬被特异性抑制。通过酵母双杂交实验，我们还发现Mec1与Atg13之间存在互作，进一步的点突变，发现Atg13上的L471-L472两个位点调控了与Mec1的结合，这两个位点的突变为能量匮乏诱导的自噬所必需。此外，我们还发现Snf1/AMPK的激酶活性调控了Mec1从线粒体上的离开。线粒体的融合机器通过影响Snf1/AMPK-Mec1的互作来影响Snf1/AMPK磷酸化Mec1，从而参与能量匮乏诱导的自噬。项目负责人在此资助下发表了5篇相关论文，其中4篇为最后通讯作者文章，受到国内外同行关注。此外还有 2 篇论文在投稿之中。在该项目资助之下，共培养 1 名博士生，3 名硕士生。

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