The Role of Highwire/DFsn Ubiquitination Complex During Synaptic Development

描述(由申请人提供)：泛素化是突触发育、信号传递和可塑性的关键调节机制。76个氨基酸多肽泛素与靶蛋白的共价结合是一种调节蛋白质稳定性、活性和定位的快速、可逆的修饰。因此，它是塑造突触的一种强有力的机制。我们已经发现了一个由E3泛素连接酶Highwire(HIW)和F-Box蛋白DFsn组成的泛素化复合体，它在控制果蝇神经肌肉接头(NMJ)的突触生长和功能方面发挥着核心作用。在哺乳动物的大脑中也发现了一个高度同源的泛素化复合体，它在调节轴突引导和突触发生方面发挥着关键作用。然而，这种泛素化复合体的分子结构和分子作用还不是很清楚。我们认为，HIW和DFsn形成一个非SCF泛素复合体，其中HIW作为E3连接酶和支架蛋白促进多亚基相互作用，不同的辅因子和泛素底物的组合赋予神经元功能时间和细胞类型特异性的调节。因此，识别这种泛素化复合体的其他成分和新的泛素靶点是理解HIW介导的泛素途径如何具体调控突触发育的关键。我们采取了两种独立的方法来解决这个问题。在生化方面，我们通过串联亲和纯化分别表达亲和标记的HIW和DFsn蛋白的苍蝇脑，鉴定了HIW/DFsn相互作用的蛋白质。研究两种HIW结合蛋白NSF和RAE1在突触发育中的作用(AIM1)，以及它们如何与HIW和DFsn一起调节泛素连接酶活性(AIM2)，将定义控制突触生长的基本泛素化机制。在基因上，我们通过HIW增强子筛选鉴定了5个HIW增强子互补组。研究这些遗传的HIW相互作用将使我们能够识别与HIW/DFsn泛素途径共同作用的其他分子途径，以塑造发育过程中形成的突触连接的结构和强度(Aim3)。

DESCRIPTION (provided by applicant): Ubiquitination is a key regulatory mechanism for synaptic development, signaling, and plasticity. The covalent attachment of the 76 aa peptide ubiquitin to target proteins is a rapid and reversible modification that regulates protein stability, activity and localization. As such, it is a potent mechanism for sculpting the synapse. We have uncovered a ubiquitination complex composed of the E3 ubiquitin ligase Highwire (Hiw) and the F-Box protein DFsn that plays a central role in controlling synaptic growth and function at the Drosophila neuromuscular junction (NMJ). A highly homologous ubiquitination complex has also been identified in the mammalian brain, where it plays a critical role in regulating axon guidance and synaptogenesis. However, the molecular architecture and molecular action of this ubiquitination complex is not well understood. We propose that Hiw and DFsn form a non-SCF ubiquitin complex where Hiw functions as an E3 ligase and a scaffolding protein to facilitate multi-subunit interaction, and the combination of different co-factors and ubiquitin substrates confers time- and cell type-specific regulation of neuronal functions. Thus identifying other components and novel ubiquitin targets of this ubiquitination complex is key to understanding how the hiw-mediated ubiquitin pathway specifically regulates synaptic development. We have taken two independent approaches to address this question. Biochemically, we identified Hiw/DFsn interacting proteins through tandem affinity purification using fly brains that express affinity-tagged Hiw and DFsn proteins, respectively. Studying the role of two of the Hiw- binding proteins, NSF and Rae1, in synaptic development (aim1), and how they work together with Hiw and DFsn to modulate the ubiquitin ligase activity (aim2) will define an essential ubiquitination machinery that controls synaptic growth. Genetically, we identified 5 hiw enhancer complementation groups through a hiw enhancer screen. Studying these genetic hiw interactors will allow us to identify other molecular pathways that work together with the Hiw/DFsn ubiquitin pathway to shape the structure and strength of synaptic connections formed during development (aim3).