Production of Human ATG Proteins for Lipidation Assays.
Production of Human ATG Proteins for Lipidation Assays.
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DOI：
10.1016/bs.mie.2016.09.055
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发表时间：
2017
影响因子：
--
通讯作者：
Schulman BA
中科院分区：
文献类型：
--
作者：
Zheng Y;Qiu Y;Gunderson JE;Schulman BA
人类在LC3和GABARAP家族中表达酵母Atg8的几个同源物，它们通过与脂类(通常是磷脂酰乙醇胺(PE))的共价连接在自噬过程中发挥关键作用，这一过程被称为脂化。Lc3和GABARAP的脂化作用调节自噬过程的许多方面，包括调节吞噬体膜的扩张，招募选定的货物进行降解，以及提供一个自噬体膜结合的平台来调节与其他调节蛋白的动态相互作用。LC3和GABARAP是一类相关的泛素样蛋白(UBL)家族(这里统称为LC3/GABARAP)，它们的脂化涉及一个发散的UBL连接级联，包括ATG7、ATG3和ATG12~ATG5-ATG16L1，分别作为E1、E2和E3酶。ATG7通过催化LC3/GABARAP的C末端腺基化和与ATG3催化的半胱氨酸的结合来启动其结合。最终，ATG12~ATG5-ATG16L1复合体催化LC3/GABARAP连接到PE或其他受体脂质上的伯氨基。本章介绍了表达和纯化人LC3或GABARAP、ATG7、ATG3和ATG12~ATG5-ATG16L1复合体的方法，用于体外研究LC3/GABARAP的脂化作用。
Humans express several orthologs of yeast Atg8, in the LC3 and GABARAP families, which play crucial roles in autophagy through their covalent ligation to lipids, typically phosphatidylethanolamine (PE), in a process known as lipidation. Lipidation of LC3 and GABARAP regulates numerous facets of the autophagy process, including regulating expansion of the phagophore membrane, recruiting selected cargoes for degradation, and providing an autophagosome membrane-bound platform mediating dynamic interactions with other regulatory proteins. LC3 and GABARAP are families of related ubiquitin-like proteins (UBLs) (referred to here collectively as LC3/GABARAP), and their lipidation involves a divergent UBL conjugation cascade including ATG7, ATG3, and ATG12~ATG5-ATG16L1 acting as E1, E2, and E3 enzymes, respectively. ATG7 initiates LC3/GABARAP conjugation by catalyzing their C-terminal adenylation and conjugation to the catalytic cysteine of ATG3. Ultimately, the ATG12~ATG5-ATG16L1 complex catalyzes LC3/GABARAP ligation to a primary amino group on PE or other acceptor lipids. This chapter describes methods for expressing and purifying human LC3 or GABARAP, ATG7, ATG3, and the ATG12~ATG5-ATG16L1 complex for in vitro studies of LC3/GABARAP lipidation.