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

描述(申请人提供)：自噬是调节真核细胞中大量蛋白质降解和细胞器周转不可或缺的过程。在自噬过程中，细胞质中的细胞器和蛋白质被吞噬到双脂双层的“自噬小体”中，在自噬小体与溶酶体融合时被大量降解。除了调节自噬的众多蛋白质外，至少15种不同的所谓“ATG”蛋白质是许多形式的自噬共同的自噬膜形成的核心成分。在这些核心成分中，有两个泛素样蛋白家族(Atg8和Atg12)，以及它们的非规范结合系统[一个非规范的E1酶(ATG7)，两个非规范的E2酶(Atg3和Atg10)，以及一个部分由UBL(Atg12~ATG5结合物，这里~指共价键)组成的非规范的E3酶]。尽管这些UBL结合级联在自噬过程中起着重要作用，并且这些途径中的缺陷与许多疾病过程有关，但我们对自噬中UBL结合的详细酶基础的了解仍然相对较少。我们建议将我们在UBL级联中的专业知识应用到自噬过程中结合UBL的非规范酶的机制和特异性上。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(目标1)和自噬UBL与其目标的连接(目标2)的潜在机制。

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).