Defining the Proteomic Composition of ER:Plasma Membrane Junctions in Brain Neurons

定义 ER 的蛋白质组组成：脑神经元的质膜连接

• 批准号: 9752682
• 负责人: James S Trimmer
• 金额: $ 19.63万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9752682
• 关键词: Adult; Alzheimer' s Disease; Biological Process; Biotinylation; Brain; Brain Diseases; Catalogs; Cell membrane; Cells; Complex; Corpus striatum structure; Data Set; Dendritic Spines; Disease; Dissection; Endoplasmic Reticulum; Event; Foundations; Functional disorder; Future; Health; Hippocampus (Brain); Hour; Huntington Disease; Intracellular Membranes; Knockout Mice; Label; Lipids; Mass Spectrum Analysis; Membrane; Membrane Proteins; Mental disorders; Methods; Molecular; Multiprotein Complexes; Neurons; Organelles; Physiological; Physiology; Play; Proteins; Proteomics; Regulation; Research Proposals; Role; Sampling; Shapes; Signal Transduction; Site; Stroke; Structure; Synapses; Time; Tissues; Voltage-Gated Potassium Channel; base; cell type; crosslink; hippocampal pyramidal neuron; in vivo; insight; mutant; nervous system disorder; neurophysiology; neuroproteomics; post stroke; protein transport; response; tandem mass spectrometry; targeted treatment; tool

Sites of contact between the endoplasmic reticulum (ER) and the plasma membrane (PM), termed ER-PM junctions or EPJs, are specialized membrane contact sites present in all cells, and at which physiologically important Ca2+ signaling events, lipid exchange, membrane protein trafficking, and other crucial cell biological processes occur. In many brain neurons, such as hippocampal pyramidal neurons (HPNs) and striatal medium spiny neurons (MSNs), EPJs represent the major Ca2+ signaling microdomain in aspiny regions of the neuron. Neuroproteomic analyses of the macromolecular signaling complexes at dendritic spines has provided information crucial to determining the specific molecular events that underlie normal synaptic signaling, and its dysregulation in neurodevelopmental and adult neurological and psychiatric disorders. A systematic dissection of the macromolecular protein complex present at EPJs at the proteomic level in any cell type, but especially in brain neurons, has not been pursued, due to the lack of appropriate methods and suitable tools. The lack of fundamental information, beginning with a molecular catalog of the protein constituents of these prominent extrasynaptic Ca2+ signaling microdomains, represents a major barrier to our understanding of basic neurophysiology and pathophysiology. We have found that an abundant and broadly expressed neuronal voltage-gated K+ channel, Kv2.1, is specifically localized to large clusters in the PM precisely at sites where EPJs form. Moreover, recent findings show that Kv2.1 actively promotes the formation and/or stabilization of EPJs through direct interaction with a resident ER protein. We propose in this exploratory research proposal to take advantage of the robust and widespread association of Kv2.1 with EPJs to undertake a concerted neuroproteomics effort to identify the protein constituents of this Ca2+ signaling microdomain in HPNs and MSNs. We will immunopurify and/or proximity label protein constituents of Kv2.1-containing EPJs in these neurons, and determine their identify by tandem mass spectrometry. These complementary neuroproteomics analyses will provide a molecular catalog of the protein constituents of these important Ca2+ signaling microdomains in HPNs and MSNs. This information will inform future studies to define the functional role of these constituents in the Ca2+ signaling events that shape the physiology and plasticity of these neurons. Lastly, as dysregulation of protein constituents of EPJs may contribute to the aberrant Ca2+ signaling that leads to degeneration of these important neurons, for example of HPNs in Alzheimer's disease and after stroke, and MSNs in Huntington's disease, they may represent important targets for therapeutic modulation.

内质网 (ER) 和质膜 (PM) 之间的接触位点，称为 ER-PM 连接点或 EPJ，是所有细胞中存在的特殊膜接触位点，在生理学上 重要的 Ca2+ 信号传导事件、脂质交换、膜蛋白运输和其他重要的细胞生物学 过程发生。在许多大脑神经元中，例如海马锥体神经元 (HPN) 和纹状体介质 在多刺神经元 (MSN) 中，EPJ 代表神经元多刺区域中主要的 Ca2+ 信号微域。 树突棘大分子信号复合物的神经蛋白质组学分析提供了 对于确定正常突触信号传导及其背后的特定分子事件至关重要的信息 神经发育和成人神经和精神疾病的失调。系统剖析 任何细胞类型中，尤其是在蛋白质组水平上，EPJ 中存在的大分子蛋白质复合物 由于缺乏适当的方法和合适的工具，大脑神经元的研究一直没有被研究。缺乏 基本信息，从这些突出的蛋白质成分的分子目录开始 突触外 Ca2+ 信号微域是我们理解基本信号的主要障碍 神经生理学和病理生理学。我们发现丰富且广泛表达的神经元 电压门控 K+ 通道 Kv2.1 专门定位于 PM 中的大簇，恰好位于以下位置： EPJ 形式。此外，最近的研究结果表明，Kv2.1 积极促进形成和/或稳定 EPJ 通过与驻留 ER 蛋白直接相互作用。我们在这个探索性研究提案中建议 利用 Kv2.1 与 EPJ 之间强大而广泛的联系来开展协调一致的行动 神经蛋白质组学努力鉴定 HPN 中 Ca2+ 信号传导微结构域的蛋白质成分， MSN。我们将在这些中对包含 Kv2.1 的 EPJ 进行免疫纯化和/或邻近标记蛋白质成分 神经元，并通过串联质谱法确定它们的身份。这些互补的神经蛋白质组学 分析将提供这些重要 Ca2+ 信号传导的蛋白质成分的分子目录 HPN 和 MSN 中的微域。这些信息将为未来的研究提供信息，以确定其功能作用 Ca2+ 信号事件中的这些成分塑造了这些神经元的生理学和可塑性。 最后，由于 EPJ 蛋白质成分的失调可能导致异常的 Ca2+ 信号传导，从而导致 这些重要神经元的退化，例如阿尔茨海默病和中风后 HPN 的退化，以及 MSNs 在亨廷顿病中可能代表治疗调节的重要靶点。