Structures/mechanisms in a noncanonical ubiquitin-like protein transfer cascade

描述（由申请人提供）：自噬是真核细胞中介导大量蛋白质降解和细胞器更新不可或缺的过程。在自噬过程中，细胞器和蛋白质被吞噬到一个双脂双层“自噬体”中，自噬体与溶酶体融合后被大量降解。除了许多调节自噬的蛋白质外，至少有15种不同的所谓的“Atg”蛋白质是许多自噬形式共同的自噬膜形成的核心成分。这些关键核心成分包括两个泛素样蛋白家族（Atg8和Atg12）及其非规范偶联系统[一个非规范E1酶（Atg7），两个非规范E2酶（Atg3和Atg10），以及一个部分由UBL组成的非规范E3酶（Atg12~Atg5偶联物，这里~指的是共价键）]。尽管这些UBL偶联级联在自噬过程中发挥了重要作用，并且这些途径中的缺陷与许多疾病过程存在关联，但我们对自噬中UBL偶联的详细酶基础的了解仍然相对初级。我们建议将我们在UBL偶联级联方面的专业知识应用于自噬过程中偶联UBL的非规范酶的机制和特异性。我们的研究计划将利用结构生物学和生物化学来了解atg7介导的自噬UBL级联的启动机制（目的1）和自噬UBL与靶标的连接机制（目的2）。

DESCRIPTION (provided by applicant): Autophagy is an indispensable process mediating bulk protein degradation and organelle turnover in eukaryotic cells. During autophagy, cytoplasmic organelles and proteins are engulfed into a double-lipid bilayer "autophagosome" to be degraded in bulk upon autophagosome fusion with a lysosome. In addition to numerous proteins regulating autophagy, at least 15 distinct so-called "Atg" proteins are core components for autophagic membrane formation common to many forms of autophagy. Among these key core components are two families of ubiquitin-like proteins (Atg8 and Atg12), and their noncanonical conjugation systems [a noncanonical E1 enzyme (Atg7), two noncanonical E2 enzymes (Atg3 and Atg10), and a noncanonical E3 enzyme partially composed of a UBL (the Atg12~Atg5 conjugate, here ~ refers to a covalent bond)]. Despite the essential roles of these UBL conjugation cascades in the process of autophagy, and the association of defects in these pathways with numerous disease processes, our knowledge of the detailed enzymatic bases for UBL conjugation in autophagy remains relatively rudimentary. We propose to apply our expertise in UBL conjugation cascades to the mechanisms and specificities of noncanonical enzymes that conjugate UBLs during autophagy. Our research plan will utilize structural biology and biochemistry to understand mechanisms underlying Atg7-mediated initiation of autophagy UBL cascades (Aim 1) and ligation of autophagy UBLs to their targets (Aim 2). PUBLIC HEALTH RELEVANCE: Autophagy is required to remove non-functional organelles and maintain protein homeostasis, and has been connected to numerous diseases, including cancers, diabetes, metabolic disorders, infections, and numerous debilitating processes associated with aging such as neurodegenerative disorders. Therefore, it is important to understand the molecular mechanisms underlying UBL conjugation in autophagy, both to provide insights into how defects in this pathway can lead to diseases, and because enzymes mediating UBL conjugation in autophagy are likely to be excellent targets for therapeutic agents.