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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

基本信息

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
项目类别：
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

项目摘要

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，但对它们的动态，