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ER-PM 连接或EPJ是所有细胞中存在的专业膜接触位点，并且在生理上 重要的Ca2+信号传导事件，脂质交换，膜蛋白运输和其他关键细胞生物学 过程发生。在许多脑神经元中，例如海马锥体神经元（HPN）和纹状体培养基 棘神经元（MSN），EPJS代表神经元的Aspiny区域中主要的Ca2+信号微域。 树突状棘的大分子信号传导复合物的神经蛋白质组学分析已提供 信息对于确定正常突触信号的基础的特定分子事件至关重要 神经发育和成人神经系统和精神疾病的失调。系统的解剖 在任何细胞类型的蛋白质组学水平上存在于EPJS的大分子蛋白复合物的 由于缺乏适当的方法和合适的工具，尚未追求脑神经元。缺乏 基本信息，从这些突出的蛋白质成分的分子目录开始 外鼻ca2+信号传导微域，代表了我们对基本的理解的主要障碍 神经生理学和病理生理学。我们发现一种丰富且广泛表达的神经元 电压门控的K+通道Kv2.1专门定位于PM中的大簇，正好在PM的位置 EPJS形式。此外，最近的发现表明，KV2.1积极促进形成和/或稳定 EPJ通过与常驻ER蛋白直接相互作用。我们在这项探索性研究建议中提出了 利用KV2.1与EPJ的强大而广泛的关联来进行一致 神经蛋白质组学的努力，以识别HPN中该Ca2+信号微域的蛋白质成分和 MSN。我们将在这些中进行免疫应激和/或接近标记的蛋白质成分 神经元，并通过串联质谱法确定其识别。这些互补的神经蛋白质组学 分析将提供这些重要CA2+信号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，以定义 这些成分在CA2+信号传导事件中塑造了这些神经元的生理和可塑性。 最后，随着EPJ的蛋白质成分的失调可能有助于引导的异常CA2+信号 要使这些重要神经元的变性，例如阿尔茨海默氏病和中风后的HPN，以及 亨廷顿氏病的MSN可能代表了治疗调节的重要靶标。