Structural and Molecular Basis of Human ATG3 Activation and Regulation for LC3 Lipid Conjugation in Autophagy

自噬中人 ATG3 激活和 LC3 脂质缀合调节的结构和分子基础

• 批准号: 10384625
• 负责人: Fang Tian
• 金额: $ 17.5万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-15 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10384625
• 关键词: ATG3 gene; Address; Autophagocytosis; Autophagosome; Bacteria; Binding; Biochemical; Biogenesis; Biological Assay; Biophysics; C-terminal; Chemicals; Clinical; Complex; Crystallization; Data; Defect; Development; Diabetes Mellitus; Disease; Enzymes; Eukaryota; Event; Geometry; Heart Diseases; Homeostasis; Human; In Situ; In Vitro; Knowledge; Lead; Lipid Bilayers; Lipids; Literature; Lysosomes; Malignant Neoplasms; Mammals; Medical; Membrane; Modality; Modeling; Molecular; Molecular Conformation; Mouse-ear Cress; Mutation; N-terminal; Nature; Nerve Degeneration; Organelles; Organism; Pathology; Pathway interactions; Patient-Focused Outcomes; Physiological; Play; Prevention; Process; Production; Proteins; Reaction; Regulation; Research; Resolution; Role; Saccharomyces cerevisiae; Side; Stress; Structural Models; Structure; System; Tube; Ubiquitin; Vacuole; Vertebral column; Virus; Yeasts; amino group; flexibility; gamma-Glutamyl Hydrolase; human disease; improved; in vivo; insight; molecular modeling; novel therapeutics; pathogen; programs; protein aggregation; recruit; therapeutic target; thioester; tumor growth

Abstract Numerous medical and scientific studies indicate that autophagy plays a key role in many diseases including neurodegenerative, infective, and cardiac diseases, and cancer. Reducing or promoting autophagic flux during particular stages of these diseases can improve patient outcomes. To understand this complex relationship between these diseases and autophagy, we must first understand what dictates its timing and regulation. Successful completion of autophagy requires a host of proteins, including Atg3. Atg3 catalyzes the conjugation of Atg8 (or LC3 in mammals) to the PE lipids in the autophagic membrane. The reaction product, Atg8-PE, acts as a marker for autophagic cargo and allows the autophagosomal membrane to be constructed. Previous studies have provided some molecular insights into this reaction; however, our understanding of its mechanism has remained remarkably elusive. In addition, Atg3's function hinges on an N-terminal amphipathic helix (NAH). This helix recognizes highly curved membranes and is required for effective Atg8-PE conjugation in vivo. In this study, we plan to examine the structural and molecular basis of human ATG3 (hATG3) activation and regulation by its interaction with the membrane for this conjugation reaction. In Aim 1, we will determine the structures and dynamics of hATG3 and its thioester intermediate hATG3-LC3. Structural models derived from high-resolution NMR will be validated using in vitro conjugation and in vivo function assays. In Aims 2 and 3, we will analyze the structural and molecular basis of ATG3 activation and regulation, and determine the molecular mechanism that drives the selective binding of ATG3's NAH to strongly curved membranes, respectively. Together, these studies will provide a mechanistic insight into hATG3 activation and regulation for the production of LC3-PE conjugate, a key molecule that triggers membrane expansion and recruits cargos for formation of the autophagosome in autophagy. Our results will contribute to a fundamental understanding of the autophagy process, which may lead to the development of new disease treatments and eventually improve the clinical patient outcome.

抽象的 大量医学和科学研究表明，自噬在许多疾病中发挥着关键作用，包括 神经退行性疾病、感染性疾病、心脏病和癌症。减少或促进自噬通量 这些疾病的特定阶段可以改善患者的治疗结果。理解这种复杂的关系 在这些疾病和自噬之间，我们首先必须了解是什么决定了它的发生时间和调控。 自噬的成功完成需要大量蛋白质，包括 Atg3。 Atg3 催化缀合 Atg8（或哺乳动物中的 LC3）与自噬膜中的 PE 脂质的结合。反应产物Atg8-PE， 作为自噬货物的标记并允许构建自噬体膜。以前的 研究为该反应提供了一些分子见解；然而，我们对其机制的理解 仍然非常难以捉摸。此外，Atg3 的功能取决于 N 端两亲性螺旋 （不）。该螺旋可识别高度弯曲的膜，并且是 Atg8-PE 有效缀合所必需的 体内。在这项研究中，我们计划检查人类 ATG3 (hATG3) 激活的结构和分子基础 并通过其与膜的相互作用来调节该缀合反应。 在目标 1 中，我们将确定 hATG3 及其硫酯中间体 hATG3-LC3 的结构和动力学。 来自高分辨率 NMR 的结构模型将使用体外缀合和体内验证 功能测定。在目标 2 和 3 中，我们将分析 ATG3 激活的结构和分子基础， 调控，并确定驱动 ATG3 的 NAH 选择性结合的分子机制 分别是强烈弯曲的膜。 总之，这些研究将为 hATG3 激活和调节提供机制见解。 LC3-PE 缀合物的生产，这是一种触发膜扩张并招募货物的关键分子 自噬过程中自噬体的形成。我们的结果将有助于对以下问题的基本理解： 自噬过程，可能会导致新疾病治疗方法的开发并最终改善 临床患者的结果。