Structural Studies of the Autophagic Ubiquitin-Like Proteins

描述（由申请人提供）：自噬-溶酶体降解机制通过去除有害的细胞器和未折叠的蛋白质聚集体等细胞质物质，有助于维持细胞稳态。这些物质的积累是人类疾病的主要原因之一，如神经变性、心脏病、癌症和感染。自噬是由称为自噬体的特殊双膜囊泡的形成引发的，自噬体将这些物质隔离并运输到溶酶体中进行降解。自噬蛋白如何产生自噬体以及细胞毒性物质如何被自噬体选择性靶向尚不清楚。提出的研究旨在利用生物化学和结构工具的组合来确定自噬体形成的关键分子机制。这些项目主要集中在自噬特异性泛素样蛋白（Ubls） at8和Atg12的机制上。这些Ubls通过控制独特的膜动力学在自噬体形成中起关键作用。在Aim 1中，我们将识别和表征E1-E2-E3级联中催化at8脂质偶联的关键相互作用。E1-E2和E2-E3通信将在生物物理层面进行研究。含有独特的E2-E3成分的分子结构将由x射线晶体学确定。在目标2中，我们将确定at8脂化的膜和底物识别机制。核磁共振和荧光光谱将用于识别的结构机制。在Aim 3中，我们将使用新开发的膜系固测定、核磁共振和生化实验来定义atg8介导的膜动力学的结构和生化要求。这些目标的结果将提供对自噬Ubls功能的机制理解，并将朝着全面描述自噬体形成迈出一大步。这项工作将不仅在自噬领域产生影响，而且将在广泛的生物学领域产生影响。

DESCRIPTION (provided by applicant): The autophagy-lysosome degradation mechanism contributes to the maintenance of cellular homeostasis by removing harmful cytoplasmic materials, such as damaged organelles and unfolded protein aggregates. The accumulation of these materials is among the primary causes of human pathologies, such as neurodegeneration, heart disease, cancer, and infection. Autophagy is initiated by the formation of specialized double-membrane vesicles termed autophagosomes, which sequester and transport such materials to lysosomes for degradation. How autophagy proteins function to generate autophagosomes and how cytotoxic materials are selectively targeted by autophagosomes are unknown. The proposed research seeks to identify key molecular mechanisms underlying autophagosome formation using a combination of biochemical and structural tools. The projects are focused on mechanisms of the autophagy-specific ubiquitin-like proteins (Ubls), Atg8 and Atg12. These Ubls play pivotal roles in autophagosome formation by controlling the unique membrane dynamics. In Aim 1, we will identify and characterize key interactions in the E1-E2-E3 cascade that catalyze the Atg8 lipid conjugation. The E1-E2 and E2-E3 communication will be studied at a biophysical level. Molecular structures containing components of the unique E2-E3 will be determined by X-ray crystallography. In Aim 2, we will determine the membrane and substrate recognition mechanism for Atg8 lipidation. NMR and fluorescence spectroscopy will used to identify structural mechanisms of the recognition. In Aim 3, we will define the structural and biochemical requirements of Atg8-mediated membrane dynamics using a newly developed membrane tethering assay, NMR, and biochemical experiments. The results from these aims will provide mechanistic understanding of the functions of autophagic Ubls and will be a large step toward comprehensive description of autophagosome formation. The impact of the work will be not only on the autophagy field but also on a broad range of biology. PUBLIC HEALTH RELEVANCE: Autophagy is the cellular degradation-recycling system that plays a crucial role in cellular homeostasis. This project seeks to understand the molecular mechanisms of autophagic ubiquitin-like proteins that mediate formation of unique vesicles called autophagosomes. The results from the research will be valuable information for development of new therapeutic strategies involving intervention into the autophagy pathway.