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

摘要 大量医学和科学研究表明，自噬在许多疾病中起着关键作用，包括 神经退行性疾病、感染性疾病、心脏疾病和癌症。减少或促进自噬通量在 这些疾病的特定阶段可以改善患者的预后。要理解这种复杂的关系 在这些疾病和自噬之间，我们必须首先理解是什么决定了它的时机和调控。 成功地完成自噬需要大量的蛋白质，包括Atg3。Atg3催化偶联反应 ATG8(或哺乳动物中的LC3)对自噬细胞膜中的PE脂类的作用。反应产物ATG8-PE， 作为自噬货物的标志，并允许构建自噬体膜。上一首 研究已经为这一反应提供了一些分子方面的见解；然而，我们对其机理的理解 仍然非常难以捉摸。此外，Atg3的S功能还依赖于N端的两亲性螺旋 (不)。这种螺旋识别高度弯曲的膜，是有效的Atg8-PE偶联所必需的 活着。在这项研究中，我们计划研究人类ATG3(HATG3)激活的结构和分子基础 以及通过它与膜的相互作用来调节这种偶联反应。 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。 从高分辨率核磁共振得到的结构模型将使用体外连接和体内验证 功能分析。在目标2和目标3中，我们将分析ATG3激活和 调节，并确定驱动ATG3的S NaH选择性结合的分子机制 分别为强弯曲膜。 总之，这些研究将为hATG3的激活和调控提供一个机械性的见解 生产LC3-PE偶联物，这是触发膜扩张和招募货物的关键分子 自噬过程中自噬小体的形成。我们的结果将有助于从根本上理解 自噬过程，这可能导致新的疾病治疗方法的开发并最终改善 患者的临床结局。

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.