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生存至关重要。我们最近的工作已经证明了泛素系统在调节肌肉神经支配（神经肌肉（NM）突触发生）和肌肉功能中的重要作用。* 泛素系统在控制蛋白质分解和运输中起着关键作用，因此影响大多数细胞功能。泛素化级联的关键组分是E3泛素连接酶，负责泛素与蛋白质的缀合，因此标记它们以进行破坏或其他命运，例如细胞内的移动/运输。我们首先从果蝇（Drosophila，dNedd 4）中分离出E3泛素连接酶Nedd 4。然后，我们发现，虽然dNedd 4（dNedd 4短，dNedd 4S）促进NM突触，较长的剪接异构体的蛋白质（dNedd 4长，dNedd 4Lo）抑制突触和损害幼虫的运动。通过对果蝇的遗传和生化分析，我们证明了dNedd 4Lo中的两个独特区域负责这些抑制作用。为了鉴定与这些独特区域相互作用的蛋白质，我们使用果蝇胚胎提取物进行了质谱分析，并鉴定了果蝇两性蛋白（dAmph）和SH 3 PX 1作为dNedd 4Lo的结合伴侣。然后，我们表明dNedd 4Lo调节肌肉T-小管中的dAmph以控制肌肉功能。已知SH 3 PX 1是一种在突触前和突触后以及在肌肉中表达的含有SH 3-PX-BAR的蛋白质，其调节神经功能。我们的初步研究结果表明，突触后dNedd 4Lo的表达导致SH 3 PX 1的消失，但dNedd 4-long如何调节SH 3 PX 1功能尚不清楚。我们假设dNedd 4Lo通过泛素化和降解SH 3 PX 1抑制突触前和突触后以及肌肉中的SH 3 PX 1功能，从而损害神经和肌肉细胞中的囊泡运输。我们的目的是破译dNedd 4Lo和SH 3 PX 1之间相互作用的生物化学和生理学性质，以及它们如何调节肌肉神经支配和功能。* 我们的具体目标是：*1）分析Nedd 4Lo介导的突触和肌肉中SH 3 PX 1稳定性的结合，泛素化和调节。2)通过分析突触超微结构，通过分析它们的遗传相互作用，比较dNedd 4Lo过表达对突触前和突触后SH 3 PX 1丢失的影响。**重要性：这些研究将描述dNedd 4蛋白和泛素化在调节肌肉神经支配和功能中的作用，这些作用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.