Mechanisms of mTOR signaling to early and late stages of autophagy

 描述（由申请人提供）：自噬是一种进化上保守的过程，通过该过程，真核细胞降解溶酶体中的细胞内细胞器和生物分子。维持细胞成分的合成、降解和再循环之间的平衡以响应营养水平和其他细胞环境的变化是重要的基本细胞过程。自噬的调节异常与衰老、先天免疫和许多人类疾病包括癌症、神经退行性疾病和免疫紊乱有关。尽管最近的进展，自噬的分子机制仍然知之甚少。我们研究的长期目标是确定自噬的机制，并了解如何利用这些知识来改善人类健康。雷帕霉素的机制靶点（mTOR），主要的生长调节激酶，是自噬诱导的关键调节因子，mTOR-自噬途径代表了自噬受损疾病中的可行靶点。在过去几年的研究中，自从我们发现ULK 1-Atg 13-FIP 200复合物作为mTOR的靶标以来，我们发现mTOR在自噬中的功能范围比以前认为的更广泛。这项更新提案的目的是定义mTOR在自噬中的扩展作用。中心假设是mTOR不仅负调节自噬的早期阶段，如omegasome/吞噬细胞形成，而且还调节吞噬细胞扩增和自噬体成熟。我们将通过追求三个具体目标来测试我们的中心假设：首先，我们将定义mTOR调节omegasome/吞噬细胞形成的途径。我们将确定mTOR介导的Atg 13磷酸化在ULK 1聚类和omegasome/吞噬细胞形成中的功能。我们将使用我们使用TALEN辅助基因组编辑技术开发的细胞系统来监测活细胞中的内源性ULK 1斑点沿着磷酸化位点突变体重建的细胞系统。其次，我们将确定ULK 1复合物调节吞噬细胞扩张的机制。我们发现MCF 2L 2，一个假定的鸟嘌呤核苷酸交换因子Rho家族GTPases，作为一个结合蛋白的Atg 13和调节Atg 9招聘。我们将确定ULK 1如何通过磷酸化调节MCF 2L 2。第三，我们将确定mTOR在自噬体成熟中的作用。我们的工作假设是mTOR和ULK 1通过磷酸化UVRAG协调调节自噬体成熟。我们将确定mTOR和ULK 1介导的UVRAG磷酸化在自噬体成熟中的功能，以及这种调节如何与自噬过程的早期阶段相协调。拟议的工作将定义mTOR在自噬中的广泛功能，这对于全面理解基本细胞过程至关重要，并为潜在目标提供关键信息以监测或操纵自噬的特定步骤。

 DESCRIPTION (provided by applicant): Autophagy is an evolutionarily-conserved process through which eukaryotic cells degrade intracellular organelles and biomolecules in the lysosome. It is the fundamental cellular process important to maintain the balance between synthesis, degradation and recycling of cellular constituents in response to changes in nutrient levels and other cellular environments. Dys-regulation of autophagy has been implicated in aging, innate immunity and many human diseases including cancers, neurodegenerative diseases, and immune disorders. Despite the recent progress, the molecular mechanisms of autophagy remain still poorly understood. The long-term goal of our study is to define the mechanisms of autophagy and understand how this knowledge can be utilized to improve human health. Mechanistic target of rapamycin (mTOR), the master growth regulatory kinase, is a key regulator of autophagy induction, and the mTOR-autophagy pathway represents a viable target in diseases where autophagy is compromised. During the past years of research since our discovery of the ULK1-Atg13-FIP200 complex as a target of mTOR, we discovered that mTOR engages in a broader range of functions in autophagy than previously thought. The objective of this renewal proposal is to define the expanded roles of mTOR in autophagy. The central hypothesis is that mTOR negatively regulates not only early stages of autophagy, such as omegasome/phagophore formation, but also phagophore expansion and autophagosome maturation. We will test our central hypothesis by pursuing three specific aims: First, we will define the pathway through which mTOR regulates omegasome/phagophore formation. We will determine the functions of mTOR-mediated phosphorylations of Atg13 in ULK1 clustering and the omegasome/phagophore formation. We will use cellular systems we developed using TALEN-assisted genome editing technique to monitor endogenous ULK1 puncta in live cells along with phosphorylation site mutant-reconstituted cellular systems. Second, we will determine the mechanism by which the ULK1 complex regulates phagophore expansion. We discovered MCF2L2, a putative guanine nucleotide exchange factor for Rho family GTPases, as a binding protein of Atg13 and a regulator of Atg9 recruitment. We will determine how ULK1 regulates MCF2L2 via phosphorylation. Third, we will determine the roles of mTOR in autophagosome maturation. Our working hypothesis is that mTOR and ULK1 coordinately regulate autophagosome maturation via phosphorylating UVRAG. We will determine the functions of mTOR- and ULK1-mediated phosphorylations of UVRAG in autophagosome maturation and how this regulation is coordinated with the early stages of autophagy processes. The proposed works will define a broad range of functions of mTOR in autophagy, which will be critical for comprehensive understanding of the fundamental cellular process and provide the key information for potential targets to monitor or manipulate specific steps of autophagy.