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

摘要 许多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、感染性疾病和心脏疾病以及癌症。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结果。为了理解这种复杂的关系 在这些疾病和自噬之间，我们必须首先了解是什么决定了它的时间和调节。 自噬的成功完成需要大量的蛋白质，包括Atg 3。Atg 3催化结合 Atg 8（或哺乳动物中的LC 3）与自噬膜中的PE脂质结合。反应产物Atg 8-PE， 作为自噬货物的标记物，并允许自噬体膜的构建。先前 研究提供了一些分子的见解，这一反应;然而，我们的理解，其机制， 仍然非常难以捉摸此外，Atg 3的功能取决于N-末端两亲性螺旋 （NAH）。该螺旋识别高度弯曲的膜，并且是在细胞中有效的Atg 8-PE缀合所需的。 vivo.在这项研究中，我们计划研究人类ATG 3（hATG 3）激活的结构和分子基础， 以及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hATG 3及其硫酯中间体hATG 3-LC 3的结构和动力学。 将使用体外偶联和体内验证源自高分辨率核磁共振的结构模型 功能测定在目标2和3中，我们将分析ATG 3激活的结构和分子基础， 调节，并确定驱动ATG 3的NAH选择性结合的分子机制， 强烈弯曲的膜，分别。 总之，这些研究将为hATG 3的激活和调节提供一个机制性的见解， LC 3-PE结合物的产生，这是一种触发膜扩张并为细胞募集货物的关键分子。 自噬中自噬体的形成。我们的研究结果将有助于从根本上了解 自噬过程，这可能会导致新的疾病治疗方法的发展，并最终改善 患者的临床结果。

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.