Analysis of synaptic protein-protein interaction by phage-display system

我们感兴趣的是哺乳动物中枢神经系统中蛋白质组成和构象的动态变化，最终导致突触结构和功能的变化。在本研究项目中，我们试图利用噬菌体展示筛选系统寻找新的突触蛋白-蛋白相互作用。结果表明，该噬菌体展示系统具有一定的实用性。它可以检测新的蛋白质相互作用，这是传统的双杂交实验理论上无法检测到的(见下文)。同样明显的是，噬菌体展示系统具有严重的局限性，使得该系统无法检测到许多已知的相互作用。综上所述，噬菌体展示系统与双杂交、亲和纯化等常规技术结合使用效果最好。我们首先利用噬菌体展示系统筛选NMDA型谷氨酸受体胞外区或L1黏附分子胞外区的结合伙伴。我们发现神经元特异性泛素连接酶fbx2是一种结合蛋白。进一步的分析表明，fbx2识别这些胞外区分子上的N-连接糖基化部分，并被认为在这些膜分子的质量控制系统中发挥了一定的作用。这一结果开辟了一种新的可能性：泛素系统在突触蛋白的调控中发挥着重要作用。为了进一步研究这种可能性，我们测试了SIGH的生物活性，SIGH是另一种泛素连接酶，通过双杂交筛选被确定为与代谢性谷氨酸受体结合的蛋白。这些分子的鉴定将有助于阐明泛素化系统精确控制突触分子数量并最终控制整体突触反应特征的新机制

We are interested in the dynamic change of protein composition and conformation at the mammalian central nervous system, which eventually leads to structural and functional change of the synapses. In this research project, we have tried to find novel synaptic protein-protein interactions using phage-display screening system. The result reveals the usefulness of the phage display system. It can detect new protein interaction which cannot be detected by conventional two-hybrid assay theoretically, (see below). It is also evident that phage-display system has severe limitations which make this system unable to detect many known interactions. As a conclusion, phage display system is most useful when used in combination with conventional techniques like two-hybrid assay or affinity purificationWe first screened binding partners for NMDA-type glutamate receptor ectodomain or L1 adhesion molecule ectodomain using phage-display system. We found neuron-specific ubiquitin ligase fbx2 as a binding protein. Further analysis reveals that fbx2 recognizes N-linked glycosylation moiety on these ectodomain molecules and is thought to have some role in the quality control system of these membrane moleculesThis result opens up a new possibility : ubiquitin system plays an important role in the control of synaptic proteins. To further examine this possibility, we tested the biological activity of sigh, another ubiquitin ligase identified as a binding protein to metabotropic glutamate receptor by two-hybrid screening. It reveals that siah actually mediates specific ubiquitination and degradation type-I metabotropic glutamate receptorIdentification of these molecules will lead to elucidate the new mechanism in which ubiquitination system precisely controls amount of synaptic molecules and eventually controls overall synaptic response characteristics