Fluorescence lifetime-based single fluorophore biosensors of post-translational modification enzyme activity

基于荧光寿命的翻译后修饰酶活性单荧光团生物传感器

• 批准号: 9359690
• 负责人: Laurie L. Parker
• 金额: $ 34.52万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9359690
• 关键词: Acetyltransferase; Advanced Development; Behavior; Binding; Biological; Biological Models; Biology; Biophysics; Biosensor; Breast Cancer Cell; Calorimetry; Cell model; Cell physiology; Cells; Complex; Cultured Cells; Cysteine; Data; Development; Disease; Energy Transfer; Environment; Enzyme Inhibitor Drugs; Enzymes; Event; Family; Fluorescence; Functional disorder; Gene Expression; Goals; Gold; Grant; Image; Imagery; Imaging technology; In Vitro; Individual; Integrin Signaling Pathway; Label; Lead; Libraries; Light; Literature; Location; Malignant Neoplasms; Maps; Measures; Methods; Microscopy; Modification; Nature; Peptides; Phosphopeptides; Phosphorylated Peptide; Phosphorylation; Phosphotransferases; Population; Post Translational Modification Analysis; Post-Translational Protein Processing; Process; Protein Kinase; Protein Tyrosine Kinase; Proteins; Proteomics; Reaction; Reader; Recombinants; Reporting; Research; Resolution; Role; Scanning; Signal Pathway; Signal Transduction; Spectrum Analysis; Stimulus; Surface Plasmon Resonance; System; Techniques; Technology; Testing; Therapeutic; Time; Titrations; United States National Institutes of Health; Validation; Work; base; cell behavior; cellular imaging; design; emergency service responder; enzyme activity; enzyme substrate; experience; experimental study; fluorescence lifetime imaging; fluorophore; imaging modality; imaging probe; innovation; insight; instrumentation; novel; novel strategies; programs; protein protein interaction; response; single cell analysis; spatiotemporal; src Homology Region 2 Domain; tool; uptake; virtual

Despite decades of research on post-translational modification enzymes, little is known about their dynamic, spatiotemporal activities on a subcellular level—and yet the nature of these subcellular activities is often key to understanding their dysfunction in cancer. The goal of this project is to develop a fluorescence lifetime-based technology to map those activities in living cells at subcellular resolution. This approach is based on robust preliminary data in which we demonstrate feasibility for detecting tyrosine kinase activity (an important PTM in many cancers) via highly resolved fluorescence lifetime shifts for three peptide probes (for Abl, Src-family, and Syk kinases), including multiplexed analysis of two probes at a time. In Aim 1, we will fully characterize the physiochemical mechanism that gives rise to the shift, which involves binding of the phosphorylated peptide product of the kinase reaction to an SH2 domain. This will allow us to understand and predict lifetime shift effects, enabling targeted probe design. In Aim 2, we will characterize and further develop the multiplexed analysis of multiple probes, using a biologically relevant model system (the integrin signaling pathway) to validate the utility of the method. In Aim 3, we will broaden the scope of the technology to detect other PTM enzyme activities, starting with Ser/Thr kinases and acetyltransferases. The work described in this proposal will deliver 1) a suite of novel, complementary probes for PTM enzyme function in cells, 2) a strategy to develop additional probes, 3) high-throughput in vitro methods for detecting PTMs via fluorescence lifetime shifts, and 4) cutting edge methods for imaging multiple probes in the same cell. These tools will make unique contributions to the understanding of PTM dynamics in the cell and to the elucidation of roles for subcellular kinase signaling in disease.

尽管对翻译后修饰酶进行了数十年的研究，但对其动态， 然而，这些亚细胞活动的性质往往是关键， 了解他们在癌症中的功能障碍。该项目的目标是开发一种基于荧光寿命的 这项技术可以在亚细胞分辨率下绘制活细胞中的这些活动。该方法基于鲁棒性 初步数据，我们证明了检测酪氨酸激酶活性的可行性（一个重要的PTM 在许多癌症中）通过三种肽探针（Abl，Src-家族， 和Syk激酶），包括一次两个探针的多重分析。在目标1中，我们将充分描述 引起转变的生理化学机制，涉及磷酸化肽的结合 激酶与SH 2结构域反应的产物。这将使我们能够理解和预测寿命的变化 影响，使有针对性的探针设计。在目标2中，我们将表征并进一步开发多路复用 多个探针的分析，使用生物学相关的模型系统（整合素信号通路）， 验证了该方法的实用性。在目的3中，我们会扩大技术的范围，以侦测其他PTM 酶活性，从Ser/Thr激酶和乙酰转移酶开始。本提案中所述的工作 将提供1）一套新的，互补的探针PTM酶功能的细胞，2）战略， 另外的探针，3）通过荧光寿命变化检测PTM的高通量体外方法，和 4)在同一细胞中成像多个探针的尖端方法。这些工具将使独特的 有助于理解PTM在细胞中的动力学和阐明亚细胞的作用， 疾病中的激酶信号传导。