Single Molecule Analysis of MAGUK Structure and Ligand Binding

这项提议的重点是突触后谷氨酸信号，它介导兴奋性 神经传递。谷氨酸受体信号是由膜相关的 鸟苷酸激酶(Maguk)是一类支架蛋白家族。脚手架决定着 通过控制神经递质受体的位置和连接进行信号转导 将它们传递到下游效应器。特别是，两个同源MAGuK(PSD-95和PSD-93) 在突触可塑性中扮演相反的角色，但它们不同活动的基础仍然是 神秘感。突触可塑性的一个关键因素是大脑皮质的翻译后修饰(PTM) 蛋白质，到目前为止发表的大多数研究中都没有这种蛋白质。 这项提案调查了PSD-95和PSD-93的翻译后调节。目标 1将检验磷酸化改变MAGuKs结构和活性的假设。我们 提出PTM位点的数量和位置的不同会导致活性的不同。 目标2将验证这样一种假设，即棕榈酰化，一种对突触至关重要的脂质修饰 靶向，改变Maguk的活动，并将它们转化为突触处观察到的细丝。 到目前为止，研究主要集中在可溶性蛋白质上，但棕榈酰化是活性所必需的。目标 3将检验MAGuKs的相变调节受体可用性的假设 有约束力的。SynGAP是一种重要的突触蛋白，可诱导PSD-95的液相分离。 这可以产生一个动态的“无膜细胞器”。我们将调查功能 相分离对受体结合和结构的影响。 我们拥有突触后唯一有效的支架相互作用的重建 并提供了对Maguk脚手架活动的唯一动力学描述。我们将使用我们独特的 解决PTM如何改变MAGUK活动的专门知识。我们开创了小说的先河 含有内在无序的蛋白质的结构精制方法 到目前为止，全长支架蛋白质的结构模型。我们将使用这些方法来 提供对Maguk结构如何影响脚手架的机械理解。我们出版的 研究表明，共同存在于PSD-95上的蛋白质具有“更高级别”的相互作用 被强迫的接近。描述兴奋信号中的分子事件是一种 神经科学中的基本挑战与大脑发育、记忆和 学习，以及许多神经和神经精神障碍。

This proposal focuses on post-synaptic glutamate signaling, which mediates excitatory neurotransmission. Glutamate receptor signaling is organized by the membrane-associated guanylate kinase (MAGuK) family of scaffold proteins. Scaffolds determine the outcome of signal transduction by controlling the location of neurotransmitter receptors and connecting them to downstream effectors. In particular, two homologous MAGuKs (PSD-95 and PSD-93) play opposing roles in synaptic plasticity yet the basis for their differential activity remains a mystery. A key factor in synaptic plasticity is post-translation modification (PTM) of the MAGuK proteins, which have been missing in most studies that have been published to date. This proposal investigates the posttranslational regulation of PSD-95 and PSD-93. Aim 1 will test the hypothesis that phosphorylation alters the structure and activity of MAGuKs. We propose that a difference in the number and location of PTM sites elicits activity differences. Aim 2 will test the hypothesis that palmitoylation, a lipid modification essential for synaptic targeting, alters MAGuK activity and coverts them into the filament observed at synapses. Studies to date focused on soluble proteins but palmitoylation is indispensable for activity. Aim 3 will test the hypothesis that phase transitions of MAGuKs regulate availability for receptor binding. SynGAP, an essential synaptic protein, induces to liquid phase separation of PSD-95. This can generate a dynamic “membrane-less organelle”. We will investigate the functional effects of phase separation on receptor binding and structure. We possess the only working reconstitution of scaffold interactions in the post-synapse and provided the only kinetic description of MAGuK scaffolding activity. We will use our unique expertise to address how PTM changes the activity of MAGuKs. We have pioneered novel methods for structural refinement of proteins containing intrinsic disorder and generated the only structural model of a full-length scaffold protein to date. We will use these approaches to provide a mechanistic understanding of how MAGuK structure affects scaffolding. Our published work suggests that proteins residing together on PSD-95 have “higher-order” interactions driven by the enforced proximity. Describing the molecular events in excitatory signaling is a fundamental challenge in neuroscience with direct relevance to brain development, memory and learning, and many neurological and neuropsychiatric disorders.