AMPylation, a novel mechanism regulating visual neurotransmission

细胞必须能够对环境的变化做出快速反应。许多此类反应的一个重要方面是对蛋白质进行快速和可逆的修饰，以调节其功能。通过激酶和磷酸酶的联合作用使蛋白质瞬间磷酸化可能是这种调控最突出的例子。最近发现的一种类似的蛋白质修饰是在蛋白质上稳定地加成单磷酸腺苷(AMP)。这一新的调控机制最初是在细菌VopS蛋白的背景下发现的，因此被称为AMP化。从那时起，细菌和真核生物的Fic结构域被发现可以使蛋白质AMPylate。果蝇基因组只编码一个Fic结构域蛋白，因此为分析AMP化的生理作用提供了显著的优势。这项提案中描述的具体目标结合了果蝇的生化和遗传方法，以确定这一机制在视觉神经传递中所起的作用。这项建议的目的是(I)使用免疫荧光和电子显微镜方法确定AMP化活性的细胞部位，(Ii)确定需要AMP化的发育阶段和细胞类型，(Iii)在果蝇视觉系统的背景下分析AMP化的生理后果，以及(Iv)结合遗传相互作用分析使用生化纯化来确定受AMP化调控的分子靶标。这些实验的完成将使我们对视觉系统中被AMP修饰的信号通路有一个全面的了解。

Cells must be able to quickly respond to changes in their environment. An important aspect of many such responses is the fast and reversible modification of proteins to regulate their function. The transient phosphorylation of proteins through the combined action of kinases and phosphatases may be the most prominent example of such regulation. A similar, more recently discovered modification of proteins is the stabile addition of adenosine 5'--‐monophosphate group (AMP) to proteins. This novel regulatory mechanism has been called AMPylation after its initial discovery in the context of the bacterial VopS protein. Since then, bacterial and eukaryotic Fic domains have been found that can AMPylate proteins. Drosophila offers a significant advantage for the analysis of the physiological role of AMPylation because fly genomes encode only a single Fic domain protein. The specific aims described in this proposal combine biochemical and genetic approaches in Drosophila to define the role that this mechanism plays in visual neurotransmission. This proposal aims (i) to determine the cellular sites of AMPylation activity using immunofluorescence and electron microscopy approaches, (ii) to define developmental stages and cell types that require AMPylation, (iii) to analyze the physiological consequences of AMPylation in the context of the Drosophila visual system, and (iv) to employ biochemical purification in conjunction with genetic interaction assays to identify the molecular targets regulated by AMPylation. Completion of these experiments will yield a comprehensive understanding of the signaling pathways in the visual system that are modified by AMPylation.