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)之间的接触部位，称为内质网-质膜 连接或EPJ是存在于所有细胞中的特化的膜接触部位，在生理上 重要的钙信号事件、脂质交换、膜蛋白运输和其他重要的细胞生物学 过程就会发生。在许多脑神经元中，如海马锥体神经元(HPN)和纹状体 刺状神经元(MSN)，EPJ代表神经元刺状区域的主要钙信号微域。 树突棘上的大分子信号复合体的神经蛋白质组分析提供了 对确定构成正常突触信号基础的特定分子事件至关重要的信息，及其 神经发育和成人神经精神障碍的调节失调。系统的剖析 在任何细胞类型的蛋白质组水平上存在于EPJ的大分子蛋白质复合体，尤其是在 由于缺乏适当的方法和工具，对脑神经元的研究一直没有得到深入的研究。缺乏 基本信息，从这些突出的蛋白质成分的分子目录开始 突触外钙信号微域，是我们理解基础的主要障碍 神经生理学和病理生理学。我们已经发现，一个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，精确地位于 EPJ表格。此外，最近的研究结果表明，Kv2.1积极地促进了 EPJ通过与驻留的ER蛋白直接相互作用。我们在这项探索性研究提案中建议 利用Kv2.1与EPJ强大而广泛的关联，进行协调一致的 神经蛋白质组学努力确定HPNS和HPNS中这个钙信号微域的蛋白质成分 MSNS。我们将免疫纯化和/或邻近标记含有Kv2.1的EPJ的蛋白成分 并通过串联质谱仪确定它们的身份。这些互补的神经蛋白质组学 分析将提供这些重要钙信号的蛋白质组成的分子目录 HPN和MSN中的微域。这些信息将为未来的研究提供信息，以定义 钙离子信号事件中的这些成分塑造了这些神经元的生理和可塑性。 最后，由于EPJ蛋白成分的失调可能导致钙信号异常，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的HPN，以及 在亨廷顿病中，MSN可能是治疗调节的重要靶点。