AMPylation, a novel mechanism regulating visual neurotransmission

描述(由申请人提供)：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将腺苷5‘-单磷酸基(AMP)稳定地加到蛋白质上。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。这些实验的完成将对视觉系统中被AMP修饰的信号通路有一个全面的了解。 公共卫生相关性：AMP化是蛋白质中稳定的腺苷5‘-单磷酸基团(AMP)的加成。最近的研究表明，这种新的调控机制在真核细胞的多种生理过程中发挥着重要作用。这一建议结合了对果蝇的生化和遗传方法，以确定这种新的机制在视觉神经传递中所起的作用。了解AMP化修饰光感受器反应的机制是重要的第一步，这样我们才能开始认识到这一新的调节层在调节健康和疾病中的视觉处理方面的潜力。

DESCRIPTION (provided by applicant): 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 is modified by AMPylation. PUBLIC HEALTH RELEVANCE: AMPylation is the stabile addition of an adenosine 5'-monophosphate group (AMP) to proteins. Recent findings have revealed that this novel regulatory mechanism plays an important role in diverse physiological process in eukaryotic cells. This proposal combines biochemical and genetic approaches in Drosophila to define the role that this novel mechanism plays in visual neurotransmission. Understanding the mechanism by which AMPylation modifies photoreceptor responses is an important first step so that we can start to appreciate the potential of this novel layer of regulation in modulating visual processing in health and disease.