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Single-molecule enzymology of protein kinases

蛋白激酶的单分子酶学

基本信息

批准号：
10353547
负责人：
Daniel J Dickinson
金额：
$ 22.55万
依托单位：
UNIVERSITY OF TEXAS AT AUSTIN
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-03-01 至 2024-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10353547
关键词：
Address Binding Biochemical Biological Biological Assay Biological Process Biomedical Research Cell physiology Cells Complement Complex Consumption Cyclic AMP-Dependent Protein Kinases Development Disease Ensure Enzymatic Biochemistry Enzymes Fluorescence Functional disorder Goals In Vitro Individual Knowledge Link Logic Malignant Neoplasms Measurement Measures Metabolic Diseases Microfluidics Microscopy Mission Modification Molecular Multiple Partners National Institute of General Medical Sciences Nature Outcome Output Phosphotransferases Post-Translational Protein Processing Protein Kinase Proteins Regulation Reporter Research Sampling Signal Transduction Signaling Protein Stimulus System Technology Testing Time Work base cell behavior developmental disease direct application enzyme activity experimental study human disease in vitro Assay in vivo innovation insight interest microfluidic technology nervous system disorder new technology novel strategies prevent protein complex reconstitution response single molecule tool

项目摘要

Project Summary / Abstract Protein kinases are ubiquitous signaling enzymes that regulate nearly every aspect of cell behavior. Kinase activity is disrupted or misregulated in countless human diseases including cancer, metabolic diseases, and neurological and developmental disorders. Kinases are regulated by upstream signals, which tune kinase enzymatic activity by means of an array of binding partners and post-translational modifications. Although in vitro reconstitution can be used to link binding partners and modifications to their effects on kinase enzymatic activity, an in vitro approach is time consuming, challenging to apply to multiple partners/modifications simultaneously, and not applicable to in vivo settings. This limitation has prevented the field from understanding how kinases integrate upstream signals to establish an appropriate level of activity. The long-term goal of the proposed research is to understand the biochemical basis of signal integration by protein kinases in vivo. To enable progress towards this goal, new experimental tools are needed because existing tools are unable to resolve the molecular state of a kinase molecule (i.e., its complement of modifications and binding partners) and simultaneously measure its activity. This project focuses on developing a single-molecule enzymatic assay for protein kinase activity. By studying single molecules, the applicants will overcome the limitations of ensemble averaging, which would otherwise preclude directly linking kinase modifications and binding partners to changes in activity because of the heterogenous nature of protein complexes in vivo. A single-molecule approach will also enable direct application to cellular protein kinases, eliminating the need for in vitro reconstitution. The central objective of this work is to gain new technical knowledge that will enable application of fluorescence-based kinase activity reporters in single molecule assays. The applicants will explore three different approaches for isolating single kinase molecules, measuring their activity and simultaneously determining which binding partners and post-translational modifications are present (Aim 1). Approaches that appear promising will then be tested on cell-derived kinase molecules to determine whether application of single-molecule kinase assays in vivo is feasible (Aim 2). The work proposed in this application is significant because it will establish the feasibility of a novel approach to measuring protein kinase activity, with the potential to ultimately yield fundamental insights into cellular signal transduction. The proposed work is innovative, in the applicant’s opinion, because it represents a fundamentally new paradigm for studying signaling by protein kinases. By establishing new tools with the potential to answer fundamental questions about signal integration by cellular protein kinases, this work will contribute to the advancement of basic biomedical research.

项目总结/摘要蛋白激酶是普遍存在的信号酶，几乎调节细胞行为的各个方面。激酶活性在无数人类疾病中被破坏或失调，包括癌症、代谢疾病和神经和发育障碍。激酶受上游信号调节，上游信号调节激酶通过结合配偶体阵列和翻译后修饰的方式来调节酶活性。虽然在体外重建可用于将结合配偶体和修饰与它们对激酶酶促活性的影响联系起来活性，体外方法是耗时的，具有挑战性的，适用于多个合作伙伴/修改同时，不适用于体内环境。这一限制使该领域无法了解激酶如何整合上游信号以建立适当的活性水平。拟议研究的长期目标是通过以下方式了解信号整合的生化基础：体内蛋白激酶。为了实现这一目标，需要新的实验工具，因为现有工具无法解析激酶分子的分子状态（即，其补充修饰和结合配偶体）并同时测量其活性。该项目的重点是开发蛋白激酶活性的单分子酶测定。通过研究单分子，申请人将克服系综平均的限制，否则将排除直接联系由于蛋白质的异质性，激酶修饰和活性变化的结合伙伴体内复合物。单分子方法也将能够直接应用于细胞蛋白激酶，消除了体外重构的需要。这项工作的中心目标是获得新的技术知识，单分子测定中基于荧光的激酶活性报告物。申请人将探索三个分离单个激酶分子，测量其活性，同时确定存在哪些结合配偶体和翻译后修饰（目的1）。的方法然后将在细胞衍生的激酶分子上进行测试，以确定是否应用体内单分子激酶测定是可行的（目的2）。本申请中提出的工作是有意义的因为它将建立一种测量蛋白激酶活性的新方法的可行性，有可能最终产生对细胞信号转导的基本见解。拟议的工作是创新，在申请人的意见，因为它代表了一个从根本上新的范式研究通过蛋白激酶传递信号。通过建立有潜力回答基本问题的新工具，关于细胞蛋白激酶的信号整合，这项工作将有助于基础研究的进展。生物医学研究