Structural Studies of the Autophagic Ubiquitin-Like Proteins

 描述（由申请人提供）：称为自噬的大量降解-再循环程序在维持细胞稳态中起关键作用。自噬的标志是重新形成被称为自噬体的双膜结合囊泡。在此过程中，被称为吞噬细胞的膜前体捕获异常的细胞质物质，如蛋白质聚集体、功能失调的细胞器和细胞内细菌，并在密封时将它们隔离到囊泡中，随后囊泡与溶酶体融合，以沉积被吞噬的内容物用于降解。这种非常不寻常的复杂细胞内膜过程需要两种专门的泛素样蛋白（Ubls），Atg 12和Atg 8。这些Ubls分别形成蛋白质-蛋白质和蛋白质-脂质缀合物，这对于适当地支撑膜和募集蛋白质因子和货物材料至关重要。该提案中的研究旨在通过在原子尺度上阐明Ubl缀合物的结构和功能来理解自噬的这些关键过程。在目标1中，我们将探索Atg 8的脂质缀合的机制。我们已经发现，Atg 8和Atg 3通过以前未描述的非共价界面相互作用。基于生化和酶动力学分析的策略将被用来证明这种相互作用的重要性，并提供新的见解的差异和相似性的Ubl~E2激活机制和典型的泛素缀合。目的2：探讨Atg 12 ~ Atg 5蛋白-蛋白偶联物共价键的作用。已知Atg 12 ~ Atg 5缀合物通过刺激Atg 8从Atg 3转移至PE来促进Atg 8脂化。我们以前的结构研究已经确定，Atg 12 ~ Atg 5的共价缀合通过稳定Atg 12和Atg 5之间的非共价界面而产生新的E3平台。然而，该平台如何实现E3-连接酶样活性仍然未知。我们产生酶-底物中间体的新策略将促进晶体学研究，最终将揭示Atg 12 ~ Atg 5在Atg 8脂化中的作用。在目标3中，我们将探索自噬膜支架组装的机制，该支架由Atg 8和Atg 12缀合物组成。新的结构的见解如何稳定吞噬细胞的这些UBL预计将出现。总的来说，从这些目标中获得的信息将产生对自噬Ubls作用的综合理解。结构信息对于合理开发靶向癌症的自噬新抑制剂特别有用。

 DESCRIPTION (provided by applicant): The bulk degradation-recycling program named autophagy plays key roles in maintenance of cellular homeostasis. The hallmark of autophagy is de novo formation of double membrane-bound vesicles called autophagosomes. During this process, the membrane precursors called phagophores capture aberrant cytoplasmic materials, such as protein aggregates, dysfunctional organelles, and intracellular bacteria, and sequester them into the vesicles upon sealing, followed by the vesicle's fusion with lysosomes for deposition of the engulfed contents for degradation. Such a highly unusual, complex process of intracellular membranes requires two specialized ubiquitin-like proteins (Ubls), Atg12 and Atg8. These Ubls form protein-protein and protein-lipid conjugates, respectively, that are crucial for properly scaffolding membranes and recruiting protein factors and cargo materials. Research in this proposal is directed toward understanding these key processes of autophagy through elucidation of the structure and functions of the Ubl conjugates at an atomic scale. In Aim 1, we will explore the mechanisms underlying the lipid conjugation of Atg8. We have found that Atg8 and Atg3 interact with each other through a previously undescribed non-covalent interface. Strategies based on biochemical and enzyme kinetic analyses will be utilized to demonstrate the importance of this interaction and provide new insights into differences and similarities of th Ubl~E2 activation mechanism and canonical ubiquitin conjugation. In Aim 2, we will explore the role of the covalent linkage of the Atg12~Atg5 protein- protein conjugate. The Atg12~Atg5 conjugate is known to promote Atg8 lipidation by stimulating the transfer of Atg8 from Atg3 to PE. Our previous structural studies have established that the covalent conjugation of Atg12~Atg5 creates a novel E3 platform through stabilization of the non-covalent interface between Atg12 and Atg5. However, how exactly this platform achieves E3-ligase like activity remains unknown. Our new strategy of generating an enzyme-substrate intermediate will facilitate crystallographic studies, which ultimately will unveil the role of Atg12~Atg5 in Atg8 lipidation. In Aim 3, we will explore the mechanisms underlying the assembly of the autophagic membrane scaffold, which consists of Atg8 and Atg12 conjugates. New structural insights into how phagophores are stabilized by these Ubls are expected to emerge. Collectively, the information that will be obtained from these aims will generate an integrated understanding of the role of autophagic Ubls. Structural information will be particularly useful for rational development of new inhibitors of autophagy targeting cancer.