Role of the ubiquitin system in Drosophila muscle function

背景：正确的肌肉发育和功能对包括苍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的分解和运输中起着关键作用，从而影响细胞中的大多数生化途径。泛素化级联中非常重要的酶是E3泛素连接酶，负责泛素与蛋白质的结合，因此标记它们进行破坏。我们从果蝇中分离到了E3泛素连接酶Nedd4，结果表明dNedd4对NM突触发生有正向调节作用。然而，我们最近发现，虽然dNedd4(dNedd4-Short)促进NM突触发生，但该蛋白的一个较长的剪接异构体(dNedd4-Long)抑制它并损害幼虫的运动。利用对果蝇的遗传和生化分析，我们证明了dNedd4-Long中的两个独特区域负责这种抑制作用。为了确定与这些独特区域相互作用的蛋白质，我们进行了质谱分析，并鉴定了几种与它们特定相关的苍蝇蛋白质，特别是果蝇两性蛋白(DAmph)。DAmph是一种突触后蛋白，在肌肉的NM连接和T管中表达，是正常肌肉功能所必需的。我们假设dNedd4Lo通过泛素化和dAmph的降解或通过改变它的运输来抑制肌肉功能。我们的目标是破译dNedd4Lo和dAmph之间相互作用的生化和生理性质，以及它们如何调节肌肉功能。我们的具体目标是：(1)分析dNedd4Lo和dAmph之间的生化相互作用。(2)研究dNedd4Lo在体内肌肉中的表达及其与dAmph的共定位。(3)研究dAmph-dNedd4LO相互作用的生理后果。这些研究将有助于描述dNedd4蛋白和泛素化在调节肌肉功能中的作用。

BACKGROUND: Proper muscle development and function are critical to animal survival, including flies. 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 biochemical pathways in the cell. Very important enzymes in the ubiquitination cascade are the E3 ubiquitin ligases, responsible for ubiquitin attachment onto proteins, therefore tagging them for destruction. We had isolated the E3 ubiquitin ligase Nedd4 from flies (Drosophila, dNedd4), and showed that dNedd4 positively regulates NM synaptogenesis. However, we recently found that while dNedd4 (dNedd4-short) promotes NM synaptogenesis, a longer splice isoform of the protein (dNedd4-long) inhibits it and impairs larval locomotion. Using genetic and biochemical analyses in flies we demonstrated that two unique regions in dNedd4-long are responsible for this inhibitory effect. To identify proteins that interact with these unique regions, we performed mass spectrometry analysis and identified several fly proteins that specifically associated with them, particularly Drosophila amphiphysin (dAmph). dAmph is a postsynaptic protein that is expressed in NM junctions and T-tubules in muscles, and is required for proper muscle function. We HYPOTHESIZE that dNedd4Lo inhibits muscle function by ubiquitination and degradation of dAmph, or by altering its trafficking. Our OBJECTIVE is to decipher the biochemical and physiological nature of interactions between dNedd4Lo and dAmph and how they regulate muscle function. Our SPECIFIC AIMS are: (1) To analyze the biochemical interactions between dNedd4Lo and dAmph. (2) To investigate in vivo muscle expression of dNedd4Lo and its co-localization with dAmph. (3) To study the physiological consequences of the dAmph-dNedd4Lo interactions. These studies will help delineate the role of dNedd4 proteins and ubiquitination in regulating muscle function.