Role of the ubiquitin system in Drosophila muscle synaptogenesis and function

泛素系统在果蝇肌肉突触发生和功能中的作用 背景：适当的肌肉发育、神经支配和功能对包括果蝇在内的动物的生存至关重要。我们最近的工作表明，泛素系统在调节果蝇肌肉神经(神经肌肉(NM)突触发生)和肌肉功能方面发挥着重要作用。 泛素系统在控制蛋白质分解和运输中起着关键作用，从而影响大多数细胞功能。泛素化级联反应的关键成分是E3泛素连接酶，负责泛素与蛋白质的结合，从而标记它们的破坏或其他命运，如细胞内的移动/运输。我们首先从果蝇(果蝇，dNedd4)中分离到E3泛素连接酶Nedd4。我们随后发现，当dNedd4(dNedd4-Short，dNedd4S)促进NM突触发生时，该蛋白的一个较长的剪接异构体(dNedd4-Long，dNedd4Lo)抑制突触发生并损害幼虫的运动。利用对果蝇的遗传和生化分析，我们证明了dNedd4Lo中的两个独特区域负责这些抑制效应。为了确定与这些独特区域相互作用的蛋白质，我们使用苍蝇胚胎提取物进行了质谱分析，并确定果蝇两栖动物蛋白(DAmph)和SH3PX1是dNedd4Lo的结合伙伴。然后我们展示了dNedd4Lo调节肌肉T管中的dAmph来控制肌肉功能。SH3PX1是一种含有SH3-PX-bar的蛋白质，在突触前、突触后和肌肉中都有表达，已知的是调节神经功能。我们的初步结果表明，突触后表达dNedd4Lo导致SH3PX1的消失，但dNedd4-Long如何调节SH3PX1功能尚不清楚。我们推测dNedd4Lo通过泛素化和降解SH3PX1在肌肉中抑制突触前和突触后的SH3PX1功能，从而损害神经和肌肉细胞中的囊泡运输。我们的目标是破译dNedd4LO和SH3PX1之间相互作用的生化和生理性质，以及它们如何调节肌肉的神经和功能。 我们的具体目标是： 1)分析Nedd4Lo介导的突触和肌肉中SH3PX1的结合、泛素化及其稳定性的调节。 2)比较突触前后dNedd4Lo过表达对SH3PX1缺失的影响，通过分析突触的遗传交互作用，分析突触超微结构。 意义：这些研究将描述dNedd4蛋白和泛素化在调节肌肉神经和功能方面的作用，这些影响对动物生命至关重要。

Role of the ubiquitin system in Drosophila muscle synaptogenesis and function BACKGROUND: Proper muscle development, innervation and function are critical to animal survival, including Drosophila. Our recent work has demonstrated an essential role for the ubiquitin system in regulating muscle innervation (neuromuscular (NM) synaptogenesis) and muscle function in flies. The ubiquitin system plays a pivotal role in controlling protein breakdown and trafficking, and thus affects most cellular functions. Key components of the ubiquitination cascade are the E3 ubiquitin ligases, responsible for ubiquitin conjugation to proteins, therefore tagging them for destruction or other fates, such as movement/traffic within cells. We first isolated the E3 ubiquitin ligase Nedd4 from flies (Drosophila, dNedd4). We then showed that while dNedd4 (dNedd4-short, dNedd4S) promotes NM synaptogenesis, a longer splice isoform of the protein (dNedd4-long, dNedd4Lo) inhibits synaptogenesis and impairs larval locomotion. Using genetic and biochemical analyses in flies we demonstrated that two unique regions in dNedd4Lo are responsible for these inhibitory effects. To identify proteins that interact with these unique regions, we performed mass spectrometry analysis using fly embryo extracts, and identified Drosophila amphiphysin (dAmph) and SH3PX1 as binding partners to dNedd4Lo. We then showed that dNedd4Lo regulates dAmph in muscle T-tubules to control muscle function. SH3PX1, a SH3-PX-BAR containing protein that is expressed both pre- and post- synaptically and in muscles, is known to regulate nerve function. Our preliminary results show that postsynaptic expression of dNedd4Lo leads to disappearance of SH3PX1, but How dNedd4-long regulates SH3PX1 function is unknown. We HYPOTHESIZE that dNedd4Lo inhibits SH3PX1 function pre and post synaptically and in muscles by ubiquitination and degradation of SH3PX1, thus impairing vesicular trafficking in nerve and muscle cells. Our OBJECTIVE is to decipher the biochemical and physiological nature of interactions between dNedd4Lo and SH3PX1 and how they regulate muscle innervation and function. Our SPECIFIC AIMS are: 1) To analyze Nedd4Lo-mediated binding to, ubiquitination of, and regulation of SH3PX1 stability in the synapse and muscle. 2) To compare the effect of dNedd4Lo overexpression to SH3PX1 loss in the pre and post synapse by analyzing synapse ultrastructure by analyzing their genetic interactions. SIGNIFICANCE: These studies will delineate the role of dNedd4 proteins and ubiquitination in regulating muscle innervation and function, effects essential for animal life.