The Autophagy Initiation Complexes

项目总结 自噬是清除内含物、受损细胞器和所有其他物质的一种保守机制。 不需要的或有害的、不能被蛋白酶体消化的物质。在人类中，自噬有主要的 在癌症、帕金森氏病、细胞内感染、神经变性和衰老中的作用。自噬涉及到 形成一种独特的杯状双层膜，即吞噬细胞，它可以膨胀，吞噬货物，以及 关闭形成自噬小体。如何将膜重塑为吞噬分子的杯状是一个 自噬中的核心和棘手问题。我们将使用结构生物学，计算机模拟，和多- 彩色3D超分辨率成像揭开杯子由来已久的谜团 杯子。自噬的启动是由ULK1/Atg1蛋白激酶的激活和靶向驱动的 和第三类磷脂酰肌醇激酶复合体(PI3KC3-C1)。该项目的具体目标 了解1)噬菌体成核，以及2)自噬前结构的调节。目标 1首先假设发现了支架蛋白Atg17的独特的S形状的几何结构 通过这个实验室，将膜的重塑驱动到杯子中。这一假说预测，在所有真核生物中， 新月体的S形状或二聚体应该作为核心自噬启动机制的一部分存在。我们将测试 假设ULK1亚单位FIP200在哺乳动物自噬中具有这种作用 显微镜、理论建模和细胞成像，包括能够可视化的3D风暴显微镜 这些细胞中的纳米大小的杯子。我们将研究ATG13、ATG101和ULK1是否以及如何在这方面进行合作 过程，以及它们在空间和时间上是如何结构化组织的。我们将研究PI3KC3-C1在 通过体外重建，电子显微镜和荧光成像的合成和 蜂窝系统。目标2将检查如何将起爆机械对准正确的位置并进行切换 在单元格中的正确时间打开。我们将描述靶向ULK1的分子和结构机制 到内质网出口部位，在那里发生饥饿诱导的吞噬体成核。我们将确定核心 控制PI3KC3-C1活性的调节电路。我们将测试VPS15蛋白激酶的假设 调节PI3KC3-C1的组装和稳定性，并随后调节Vps34激酶的活性 变构和非催化方式。使用冷冻-EM、质谱学、生物化学和细胞成像，我们将 确定该电路是否以及如何用于ULK1对PI3KC3-C1活性的调节 通过蛋白NRBF2、Bcl2和AMBRA1的结合而被磷酸化。我们还将探索 假设PI3KC3可以通过BECN1 Bara结构域在膜结合水平上受到调节， 以PI3KC3-C2-Rubcon络合物为模型体系。

PROJECT SUMMARY Autophagy is a conserved mechanism for the clearance of inclusions, damaged organelles, and all other unneeded or harmful materials that cannot be digested by proteasomes. In humans, autophagy has major roles in cancer, Parkinson’s disease, intracellular infection, neurodegeneration, and aging. Autophagy involves the formation of a unique cup-shaped double membrane, the phagophore, which expands, engulfs cargo, and closes to form the autophagosome. How membranes are remodeled into the cup shape of the phagophore is a central and intractable question in autophagy. We will use structural biology, computer simulations, and multi- color 3D superresolution imaging of the cup to unravel the long-standing mystery of the origin and shaping of the cup. Autophagy initiation is driven by the activation and targeting of the ULK1/Atg1 protein kinase complex, and the class III phosphatidylinositol kinase complex I (PI3KC3-C1). The specific aims of the project are to understand 1) nucleation of the phagophore, and 2) regulation of the pre-autophagosomal structure. Aim 1 begins with the hypothesis that the unique S-shaped geometry of the scaffolding protein Atg17, discovered by this laboratory, drives remodeling of membrane into cups. This hypothesis predicts that in all eukaryotes, an S-shape or dimer of crescents should exist as part of the core autophagy initiation machinery. We will test the hypothesis that ULK1 subunit FIP200 has this role in mammalian autophagy using x-ray diffraction, electron microscopy, theoretical modeling, and cell imaging, including 3D STORM microscopy capable of visualizing these nanoscale cups in cells. We will work out whether and how ATG13, ATG101, and ULK1 cooperate in this process, and how they are organized structurally in space and time. We will examine the role of PI3KC3-C1 in cup formation through in vitro reconstitution, electron microscopy, and fluoresence imaging of synthetic and cellular systems. Aim 2 will examine how the initiation machinery is targeted to the right location and switched on at the correct time in cells. We will characterize the molecular and structural mechanisms for targeting ULK1 to ER exit sites, where starvation-induced phagophore nucleation occurs. We will determine the core regulatory circuitry that controls PI3KC3-C1 activity. We will test the hypothesis that the VPS15 protein kinase regulates the assembly and stability of PI3KC3-C1, and subsequently regulates VPS34 kinase activity in an allosteric and non-catalytic manner. Using cryo-EM, mass spectrometry, biochemistry, and cell imaging, we will determine whether and how this circuitry is used in the regulation of PI3KC3-C1 activity by ULK1 phosphorylation and by the binding of the proteins NRBF2, Bcl-2, and AMBRA1. We will also explore the hypothesis that PI3KC3 can be regulated at the level of membrane binding through the BECN1 BARA domain, using as a model system the PI3KC3-C2-Rubicon complex.