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

描述（由申请人提供）：自噬是介导真核细胞中大量蛋白质降解和细胞器更新的不可或缺的过程。在自噬过程中，细胞质细胞器和蛋白质被吞入双脂质双层“自噬体”中，在自噬体与溶酶体融合后大量降解。除了众多调节自噬的蛋白质之外，至少有 15 种不同的所谓“Atg”蛋白质是许多形式的自噬中常见的自噬膜形成的核心成分。这些关键的核心成分包括两个泛素样蛋白家族（Atg8 和 Atg12）及其非规范缀合系统 [一种非规范 E1 酶（Atg7）、两种非规范 E2 酶（Atg3 和 Atg10）和一种部分由 UBL 组成的非规范 E3 酶（Atg12~Atg5 缀合物，这里 ~ 指共价键）]。尽管这些 UBL 结合级联在自噬过程中发挥着重要作用，并且这些途径中的缺陷与许多疾病过程相关，但我们对自噬中 UBL 结合的详细酶学基础的了解仍然相对初级。我们建议将我们在 UBL 缀合级联方面的专业知识应用于自噬过程中缀合 UBL 的非经典酶的机制和特异性。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 级联启动（目标 1）以及自噬 UBL 与其靶点连接的潜在机制（目标 2）。 公共健康相关性：自噬是去除无功能细胞器和维持蛋白质稳态所必需的，并且与许多疾病有关，包括癌症、糖尿病、代谢紊乱、感染以及许多与衰老相关的衰弱过程，例如神经退行性疾病。因此，了解自噬中 UBL 结合的分子机制非常重要，这不仅可以深入了解该途径的缺陷如何导致疾病，而且因为介导自噬中 UBL 结合的酶可能是治疗药物的绝佳靶标。

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.