Activity-dependent probes for spatially-defined proteomics

用于空间定义的蛋白质组学的活性依赖性探针

• 批准号: 10686705
• 负责人: Christina Kim
• 金额: $ 141.43万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10686705
• 关键词: Alzheimer' s Disease; Animal Behavior; Behavioral; Benchmarking; Biochemical; Biological; Biological Markers; Brain; Brain Diseases; Cells; Communities; Cultured Cells; Disease model; Enzymes; Epilepsy; Gene Expression; Genetic; Genome; Goals; Hallucinogens; Health; Human; Label; Location; Methodology; Molecular; Mus; Neurons; Neurophysiology - biologic function; Neurosciences; Pattern; Pharmaceutical Preparations; Population; Property; Protein Engineering; Proteins; Proteome; Proteomics; Research; Spatial Distribution; Subgroup; Technology; Testing; Therapeutic; Translating; autism spectrum disorder; drug development; drug discovery; experience; high throughput screening; improved; insight; link protein; liquid chromatography mass spectrometry; neural; neuropsychiatric disorder; new therapeutic target; programs; protein expression; response; transcriptome

Project Summary Uncovering the molecular properties of functionally-defined neural ensembles is essential for understanding how these networks give rise to circuit function and animal behavior. This proposal aims to develop and deploy new molecular technologies that will enable the sub-cellular tagging and enrichment of proteins in activated subgroups of neurons in the brain. Linking protein expression to neural function at a large and unbiased scale is currently not possible with existing technologies. Thus the research program proposed here will fill a critical unmet gap in the molecular toolbox for neuroscientists. While prior technologies have focused on gaining genetic access to the genome or transcriptome of activated neurons, the approach here will identify the actual proteins expressed in specific subcellular compartments of functionally-relevant neurons. This is a key distinction, as gene expression alone cannot reveal to the actual physical location and expression patterns of translated proteins – which are the ultimate molecules that carry out the specific biochemical functions of our cells. To enable this goal, new activity-dependent proximity labeling probes will be developed using protein engineering. These probes will be improved and optimized through high-throughput screens performed in cultured cells, and then adapted for use in mammalian neurons. Concurrently, the probes will be tested and characterized in the mouse brain to improve and benchmark their function. To demonstrate their utility, the activity-dependent probes will be used to tag the proteins that are present in neurons undergoing high neural activity in response to a behavioral drug experience in mice. 5-MeO-DMT is a hallucinogenic drug that in humans has been associated with therapeutic potential for treating neuropsychiatric diseases. Neurons activated by 5-MeO-DMT will be labeled by the activity-dependent probes, and their spatial distribution throughout the brain will be examined using the fluorescent read-out of the new molecular enzyme. In addition, the probes will also tag the proteins that are present in these activated neurons, allowing the enrichment and unbiased profiling of these molecules using liquid chromatography mass spectrometry (proteomics). The sub- cellular proteome of these neurons will provide essential biological insight into the mechanism of hallucinogenic drugs, in addition to providing potential downstream targets for new drug discovery and development. More broadly, the new probes will be distributed freely to the neuroscience community to enable the study of protein expression in functionally-relevant populations of neurons.

项目概要 揭示功能定义的神经元集群的分子特性对于理解至关重要 这些网络如何产生电路功能和动物行为。该提案旨在开发和部署 新的分子技术将能够对活化的蛋白质进行亚细胞标记和富集 大脑中的神经元亚群。在大规模且无偏见的范围内将蛋白质表达与神经功能联系起来 目前现有技术还无法实现。因此，这里提出的研究计划将填补一个关键的空缺。 神经科学家分子工具箱中未满足的空白。虽然现有技术的重点是获得 通过基因获取激活神经元的基因组或转录组，这里的方法将识别实际的 在功能相关神经元的特定亚细胞区室中表达的蛋白质。这是一把钥匙 区别，因为基因表达本身无法揭示实际的物理位置和表达模式 翻译蛋白质——它们是执行我们的特定生化功能的最终分子 细胞。为了实现这一目标，将使用蛋白质开发新的活性依赖性邻近标记探针 工程。这些探针将通过在 培养细胞，然后适用于哺乳动物神经元。同时，将测试探针并 对小鼠大脑进行表征，以改善和基准其功能。为了展示它们的实用性， 活动依赖性探针将用于标记存在于经历高神经元的神经元中的蛋白质 对小鼠行为药物体验的反应活动。 5-MeO-DMT 是一种致幻药物， 人类具有治疗神经精神疾病的潜力。神经元 被 5-MeO-DMT 激活的探针将被活性依赖性探针标记，并且它们的空间分布 整个大脑将使用新分子酶的荧光读数进行检查。此外， 探针还将标记这些激活的神经元中存在的蛋白质，从而实现富集和 使用液相色谱质谱法（蛋白质组学）对这些分子进行无偏分析。子 这些神经元的细胞蛋白质组将为了解其机制提供重要的生物学见解 致幻药物，除了为新药发现和提供潜在的下游靶点 发展。更广泛地说，新的探针将免费分发给神经科学界，以实现 研究功能相关的神经元群体中的蛋白质表达。