The Autophagy Initiation Complexes

自噬起始复合物

• 批准号: 9982076
• 负责人: James H Hurley
• 金额: $ 29.81万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-15 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9982076
• 关键词: 1-Phosphatidylinositol 3-Kinase; 3-Dimensional; Aging; Autophagocytosis; Autophagosome; BCL2 gene; Binding; Binding Proteins; Biochemical; Biochemistry; Biological; Biological Models; Biophysics; Cell Survival; Cells; Color; Complex; Computer Simulation; Cryoelectron Microscopy; Disease; Electron Microscopy; Enzymes; Estrogen receptor positive; Eukaryota; Geometry; Human; Image; In Vitro; Infection; Laboratories; Lipids; Location; Lysosomes; Malignant Neoplasms; Mammals; Mass Spectrum Analysis; Membrane; Microscopy; Modeling; Molecular Structure; Nerve Degeneration; Neurodegenerative Disorders; Organelles; Outcome; Parkinson Disease; Phosphatidylinositols; Phosphorylation; Phosphotransferases; Process; Protein Dynamics; Protein Engineering; Protein Kinase; Regulation; Resolution; Role; Scaffolding Protein; Shapes; Signal Transduction; Site; Starvation; Stress; Structure; System; Testing; Theoretical model; Thermodynamics; Time; Vps15 protein kinase; Work; X ray diffraction analysis; Yeasts; cellular imaging; dimer; innovation; multicatalytic endopeptidase complex; nanoscale; novel; protein complex; protein structure; reconstitution; response; scaffold; structural biology; unilamellar vesicle

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.

项目概要 自噬是一种保守的机制，用于清除内含物、受损细胞器和所有其他细胞器 蛋白酶体无法消化的不需要的或有害的物质，在人类中，自噬具有重要作用。 自噬在癌症、帕金森病、细胞内感染、神经退行性疾病和衰老中的作用。 形成独特的杯形双层膜，即吞噬细胞，它会膨胀、吞没货物，并且 关闭形成自噬体是如何将膜重塑为吞噬细胞的杯形的。 我们将利用结构生物学、计算机模拟和多重方法来解决自噬中的核心和棘手问题。 杯子的彩色3D超分辨率成像，解开长期以来的杯子起源和塑造之谜 自噬的启动是由 ULK1/Atg1 蛋白激酶的激活和靶向驱动的。 复合物，以及 III 类磷脂酰肌醇激酶复合物 I (PI3KC3-C1) 该项目的具体目标。 目的是了解 1) 吞噬细胞的成核，以及 2) 自噬体前结构的调节。 1 首先假设支架蛋白 Atg17 具有独特的 S 形几何形状，被发现 该实验室推动膜重塑为杯状结构，该假设预测在所有真核生物中， S 形或新月体二聚体应作为核心自噬启动机制的一部分存在。 使用 X 射线衍射、电子学等假设 ULK1 亚基 FIP200 在哺乳动物自噬中具有这种作用 显微镜、理论模型和细胞成像，包括能够可视化的 3D STORM 显微镜 我们将研究 ATG13、ATG101 和 ULK1 在这方面是否以及如何合作。 过程，以及它们在空间和时间上的结构组织方式，我们将研究 PI3KC3-C1 在其中的作用。 通过体外重建、电子显微镜和合成和荧光成像来形成杯 目标 2 将检查启动机制如何定位到正确的位置并进行切换。 我们将描述靶向 ULK1 的分子和结构机制。 到内质网出口位点，饥饿诱导的吞噬细胞成核发生的地方我们将确定核心。 我们将检验 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 激酶活性 使用冷冻电镜、质谱、生物化学和细胞成像，我们将采用变构和非催化方式。 确定该电路是否以及如何用于 ULK1 调节 PI3KC3-C1 活动 我们还将探索 NRBF2、Bcl-2 和 AMBRA1 蛋白的磷酸化和结合。 假设 PI3KC3 可以通过 BECN1 BARA 结构域在膜结合水平上进行调节， 使用 PI3KC3-C2-Rubicon 复合体作为模型系统。