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

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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.