Single Molecule Analysis of MAGUK Structure and Ligand Binding

MAGUK 结构和配体结合的单分子分析

• 批准号: 9884794
• 负责人: Mark E Bowen
• 金额: $ 44.49万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-01-10 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9884794
• 关键词: Active Sites; Address; Affect; Affinity; Allosteric Regulation; Award; Binding; Brain; Clinical; Cytoplasmic Tail; Data; Deposition; Development; Disease; Epilepsy; Event; Family; Filament; Future; Genetic; Glutamate Receptor; Glutamates; In Vitro; Individual; Kinetics; Knowledge; Learning; Length; Ligand Binding; Ligands; Link; Lipids; Liquid substance; Location; Mediating; Membrane; Memory; Mental disorders; Methods; Modification; Molecular; Molecular Conformation; Mutation; Nerve Degeneration; Neurosciences; Neurotransmitter Receptor; Oils; Organelles; Outcome; Peptides; Phase; Phase Transition; Phosphorylation; Phosphotransferases; Physiological; Play; Post-Translational Protein Processing; Post-Translational Regulation; Protein Isoforms; Protein Kinase; Proteins; Proteomics; Publishing; Reaction; Receptor Signaling; Role; Scaffolding Protein; Schizophrenia; Sequence Homology; Signal Transduction; Signaling Protein; Site; Specificity; Stroke; Structural Models; Structure; Synapses; Synaptic plasticity; System; Testing; Translations; Water; Work; autism spectrum disorder; druggable target; glutamatergic signaling; in vivo; interest; membrane activity; membrane-associated guanylate kinase; nervous system disorder; neuropsychiatric disorder; neurotransmission; novel; palmitoylation; particle; post stroke; receptor; receptor binding; reconstitution; recruit; scaffold; single molecule

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.

该提案重点关注突触后谷氨酸信号传导，它介导兴奋性 神经传递。谷氨酸受体信号传导是由膜相关的 鸟苷酸激酶 (MAGuK) 支架蛋白家族。支架决定结果 通过控制神经递质受体的位置和连接来进行信号转导 将它们传递给下游效应器。特别是，两种同源 MAGuK（PSD-95 和 PSD-93） 在突触可塑性中发挥相反的作用，但它们差异活动的基础仍然是 神秘。突触可塑性的一个关键因素是 MAGuK 的翻译后修饰 (PTM) 迄今为止已发表的大多数研究中都缺少蛋白质。 该提案研究了 PSD-95 和 PSD-93 的翻译后调控。目的 图 1 将检验磷酸化改变 MAGuK 的结构和活性的假设。我们 认为 PTM 位点数量和位置的差异会引起活性差异。 目标 2 将检验以下假设：棕榈酰化（一种突触必需的脂质修饰） 靶向、改变 MAGuK 活性并将其转化为在突触处观察到的细丝。 迄今为止的研究主要集中在可溶性蛋白质上，但棕榈酰化对于活性是必不可少的。目的 3 将检验 MAGuK 的相变调节受体可用性的假设 绑定。 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。 我们拥有突触后支架相互作用的唯一有效重建 并提供了 MAGuK 支架活性的唯一动力学描述。我们将用我们独特的 解决 PTM 如何改变 MAGuK 活动的专业知识。我们开创了小说 对含有内在无序的蛋白质进行结构精炼的方法，并产生了唯一的 迄今为止全长支架蛋白的结构模型。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。我们发表的 研究表明，PSD-95 上共同存在的蛋白质具有“高阶”相互作用驱动 通过强制接近。描述兴奋信号传导中的分子事件是 神经科学中与大脑发育、记忆和记忆直接相关的基本挑战 学习以及许多神经系统和神经精神疾病。