营养感受激酶mTORC1对自噬特异的脂质磷酸化激酶的关键性调控机制研究

Autophagy is a highly conserved catabolic process that clears damaged proteins and organelles within the cell. This catabolic pathway is often activated in cancer cells in response to multiple stresses including hypoxia and nutrient limitation. The activation of autophagy is associated with tumor initiation, progression, microenvironment, and drug resistance. The class III phosphatidylinositol 3-kinase (PI3KC3) complex plays a central role in autophagosome biogenesis by producing phosphatidylinositol 3-phosphate (PI3P) on the initiating phagophore membrane to recruit and nucleate membrane associate proteins for membrane extension. Along with other groups, we demonstrated that p150-Vps34-Beclin 1 constitutes the core PI3KC3 complex and this complex is recruited to autophagosome/phagophore membrane by ATG14, or to endosome membrane by UVRAG and Rubicon. NRBF2 is the fifth subunit of the autophagic PI3KC3 complex. NRBF2 directly binds to ATG14 and promotes the complex assembly. PI3KC3 activity on autophagosomes is robustly induced by nutrient starvation. However, it is still unclear how starvation signal is transduced to the autophagy machinery. We recently found that NRBF2 is phosphorylated by mTORC1, the key nutrient sensing kinase, and this phosphorylation event plays a fine-tune regulatory role in autophagy. In this project we aim to dissect how NRBF2 phosphorylation is delicately regulated by upstream nutrient sensing kinases in autophagy and tumorigenesis through genetic and biochemical approaches. These data will provide new insights into the regulatory mechanism of mammalian autophagy and the molecules involved might serve as new drug targets for cancer.

细胞自噬是广泛存在于细胞中的一种可通过自噬体包裹，溶酶体降解途径清除胞质蛋白和细胞器的代谢途径。因为自噬在肿瘤的发生发展微环境及抗药性中起重大作用，其调控机制在近年来引起了研究者们的广泛关注。自噬通路主要被营养感受蛋白激酶mTORC1抑制调控，但我们mTORC1在自噬中的直接底物知之甚少。进化保守的脂质蛋白激酶PI3KC3在动物细胞自噬起关键作用。我们最近的研究表明NRBF2, 自噬特异的PI3KC3复合体五个核心成员之一，可能是mTORC1的直接底物。我们进一步推测mTORC1介导的NRBF2磷酸化是一个调控自噬的精准开关。本课题将通过在细胞和体外重组蛋白实验系统中研究mTORC1所致NRBF2的磷酸化对PI3KC3复合物形成、脂质激酶活性及自噬水平的影响。借助对NRBF2磷酸化及自噬的精准调控，我们将在胰腺癌及三阴乳腺癌癌症模型中进一步探索自噬对肿瘤发生发展的影响。

本项目围绕PI3KC3C1复合物激酶活性的调控机制开展研究。我们通过体外纯化蛋白复合物证实了NRBF2确实为五元PI3KC3-C1复合物的一个组分。我们在体外酶活实验中证实了NRBF2能促进PI3KC3-C1复合物的激酶活性，包含持续去磷酸化NRBF2-AA突变体的PI3KC3-C1复合物具有最高的酶活。NRBF2促进PI3KC3-C1酶活的机制可能是由于NRBF2的存在增加了PI3KC3-C1复合物的膜桥连能力。另一方面，我们自主研发了PI3KC3-C1激酶体外高通量活性检测方法。运用新建立的体外高通量活性检测方法,我们从激酶库中初步筛选获得若干个激活PI3KC3-C1的小分子。进一步细胞实验筛选后，我们获得了激活PI3KC3-C1复合物的三个小分子Vx-11e，PI-103以及Dinaciclib。通过一系列细胞实验，我们明确了Vx-11e对自噬的影响，并初步解析了其调控自噬的机制。同时我们还发现Vx-11e对于引发ALS疾病的毒性蛋白聚集物TDP43具有显著的降解效果。我们将在ALS疾病小鼠模型中验证该小分子对ALS疾病的干预作用。此项研究为自噬信号通路研究以及ALS疾病的干预提供新思路。