Autophagy initiation by the Atg1 complex

Atg1 复合物启动自噬

• 批准号: 9120391
• 负责人: James H Hurley
• 金额: $ 29.83万
• 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 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9120391
• 关键词: Aging; Architecture; Area; Autophagocytosis; Autophagosome; Binding; Biochemical; Biogenesis; Biological Assay; Biological Models; C-terminal; Cell Survival; Cell physiology; Cells; Complex; Coupled; Cytosol; Deuterium; Disease; Electron Microscopy; Engineering; Eukaryota; Event; Health; Homeostasis; Human; Hydrogen; In Vitro; Infection; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Membrane; Membrane Biology; Membrane Proteins; Modeling; Monitor; Mutagenesis; Mutate; Neurodegenerative Disorders; Organelles; Outcome; Parkinson Disease; Pathway interactions; Phosphorylation; Protein Dephosphorylation; Regulation; Research; Role; Scanning; Shapes; Signal Transduction; Staging; Starvation; Stress; Structure; Testing; Tissues; Vesicle; Yeasts; base; feeding; in vitro Assay; in vivo; insight; mutant; novel; protein complex; protein expression; reconstitution; research study; response; scaffold; sensor; therapeutic development; therapeutic target; three dimensional structure

DESCRIPTION (provided by applicant): Autophagy is a conserved pathway for cell survival during starvation and for clearance of damaged, toxic, or excess organelles and inclusions from the cell. Bulk cytosol, organelles, and other autophagic cargo are taken up within a double membrane vesicle known as the autophagosome. Autophagy is central to cell function and human health, yet the physical basis of autophagosome biogenesis is almost completely unknown. The proposed research will yield, in near-atomistic detail, the mechanism for the earliest stage in autophagosome biogenesis. We hypothesize that the key event in the initiation of the double membrane of the autophagosome is the clustering of high curvature vesicles containing the membrane protein Atg9 at the preautophagosomal structure (PAS). This event is orchestrated by the Atg1 complex, which in yeast consists of Atg1, Atg13, Atg17, Atg29, and Atg31. The latter three comprise the Atg17-Atg31-Atg29 subcomplex, which is the first to arrive at the PAS. We determined the structure of the Atg17-Atg31-Atg29 complex, revealing a remarkable S-shaped double crescent and suggesting a model for vesicle scaffolding. We also discovered that the C-terminal early autophagy targeting/tethering (EAT) domain of Atg1 is a potent sensor for high membrane curvature and tethers high curvature vesicles. These insights led us to a detailed hypothesis for the scaffolding and tethering of the vesicles that initiate autophagosome biogenesis. The specific aims of this project are as follows: 1. we will understand how the Atg1 EAT domain tethers highly curved vesicles. Tethering will be assayed in vitro and correlated with function in yeast cells. The structural basis for tethering will be determined using scanning mutagenesis and hydrogen-deuterium exchange coupled to mass spectrometry. 2. The three dimensional structure of the Atg1 complex will be determined by SAXS and EM, and structural interfaces mapped by scanning mutagenesis to develop a pseudo-atomic model for the complex. 3. In a stringent test of the central hypothesis, an Atg9 lipopeptide based model for precursor vesicles will be used to reconstitute the early PAS in vitro. The model system will be used to probe whether Atg13 dephosphorylation during starvation triggers the assembly of a tethering-competent form of the Atg1 complex. Together, these aims will flesh out the main early events of autophagy initiation in mechanistic and structural detail.

描述（由申请人提供）：自噬是饥饿期间细胞存活以及从细胞中清除受损、有毒或过量的细胞器和内含物的保守途径。大量胞质溶胶、细胞器和其他自噬货物被吸收在称为自噬体的双层膜囊泡中。自噬对于细胞功能和人类健康至关重要，但自噬体生物发生的物理基础几乎完全未知。拟议的研究将以接近原子的细节产生自噬体生物发生最早阶段的机制。我们假设自噬体双层膜启动的关键事件是含有膜蛋白 Atg9 的高曲率囊泡在自噬体前结构 (PAS) 处聚集。该事件由 Atg1 复合体精心策划，在酵母中该复合体由 Atg1、Atg13、Atg17、Atg29 和 Atg31 组成。后三个组成了 Atg17-Atg31-Atg29 子复合体，它是第一个到达 PAS 的。我们确定了 Atg17-Atg31-Atg29 复合物的结构，揭示了显着的 S 形双新月形，并提出了囊泡支架的模型。我们还发现 Atg1 的 C 端早期自噬靶向/束缚 (EAT) 结构域是高膜曲率和束缚高曲率囊泡的有效传感器。这些见解使我们对启动自噬体生物发生的囊泡的支架和束缚产生了详细的假设。该项目的具体目标如下： 1.我们将了解Atg1 EAT结构域如何束缚高度弯曲的囊泡。束缚将在体外进行测定，并与酵母细胞的功能相关联。束缚的结构基础将通过扫描诱变和氢-氘交换与质谱联用来确定。 2. Atg1复合物的三维结构将通过SAXS和EM确定，并通过扫描诱变映射结构界面，以开发该复合物的伪原子模型。 3. 在对中心假设的严格测试中，基于 Atg9 脂肽的前体囊泡模型将用于在体外重建早期 PAS。该模型系统将用于探测饥饿期间 Atg13 去磷酸化是否会触发 Atg1 复合物的束缚形式的组装。这些目标将共同充实自噬启动的主要早期事件的机制和结构细节。