Elucidation of the mode of action of Atg12-Atg5 conjugate in autophagy

自噬是一个整体降解系统，它涉及到一个被称为自噬小体的双层膜小泡的形成。自噬小体的形成需要两个泛素样蛋白：Atg8和Atg12。它们分别通过与E1和E2酶的一系列酶反应与磷脂酰乙醇胺(PE)和ATG5结合。在本研究中，我们阐明了Atg12-ATG5作为E3酶在Atg8-PE偶联反应中的作用方式。我们建立了一种基于结构信息的生化分析方法来确定Atg8-PE偶联反应中的一种E2酶Atg3的催化中心构型。这一方法表明，Atg12-ATG5偶联物诱导了Atg3催化中心的构象变化，从而增强了其E2活性。此外，突变分析表明，在没有Atg12-ATG5结合物的情况下，Atg3的活性是如何受到抑制的。我们正在尝试分析Atg12-ATG5共轭/Atg3络合物的晶体结构。

Autophagy is a bulk degradation system, which involves the formation of a double-membrane vesicle called autophagosome. Autophagosome formation requires two ubiquitin-like proteins, Atg8 and Atg12. They are conjugated to the phosphatidylethanolamine (PE) and Atg5, respectively, via a series of enzymatic reactions with E1 and E2 enzyme. In this study, we elucidated the mode of action of Atg12-Atg5 as an E3 enzyme in the Atg8-PE conjugation reaction. We established a biochemical assay based on the structural information to determine the configuration of the catalytic center of Atg3, which is an E2 enzyme in Atg8-PE conjugation reaction. This approach revealed that Atg12-Atg5 conjugate induces a conformational change in the catalytic center of Atg3 to enhance its E2 activity. Moreover, mutational analyses indicated how the activity of Atg3 is suppressed in the absence of Atg12-Atg5 conjugate. We are attempting to analyze the crystal structure of Atg12-Atg5 conjugate/Atg3 complex.