The Role of Highwire/DFsn Ubiquitination Complex During Synaptic Development

描述（申请者提供）：泛素化是突触发育、信号传导和可塑性的关键调控机制。76aa肽泛素与靶蛋白的共价附着是一种快速、可逆的修饰，可调节蛋白质的稳定性、活性和定位。因此，它是一种雕刻突触的有效机制。我们发现了一个由E3泛素连接酶Highwire （Hiw）和F-Box蛋白DFsn组成的泛素化复合物，它在控制果蝇神经肌肉连接处（NMJ）的突触生长和功能中起核心作用。一个高度同源的泛素化复合体也在哺乳动物大脑中被发现，它在调节轴突引导和突触发生中起着关键作用。然而，这种泛素化复合物的分子结构和分子作用尚不清楚。我们提出Hiw和DFsn形成一个非scf泛素复合物，其中Hiw作为E3连接酶和支架蛋白促进多亚基相互作用，不同辅助因子和泛素底物的组合赋予神经元功能的时间和细胞类型特异性调节。因此，识别这种泛素化复合物的其他成分和新的泛素靶点是理解低介导的泛素通路如何特异性调节突触发育的关键。我们采取了两种独立的方法来解决这个问题。生物化学方面，我们利用分别表达亲和标记Hiw和DFsn蛋白的果蝇大脑，通过串联亲和纯化鉴定了Hiw/DFsn相互作用蛋白。研究两种Hiw结合蛋白NSF和Rae1在突触发育（aim1）中的作用，以及它们如何与Hiw和DFsn一起调节泛素连接酶活性（aim2），将确定控制突触生长的基本泛素化机制。遗传学上，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hiw相互作用将使我们能够确定与hiw /DFsn泛素途径一起工作的其他分子途径，以塑造发育过程中形成的突触连接的结构和强度（目标3）。

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).