Development of imaging reagents to monitor GTP levels and GTP/GDP ratios in vivo

开发成像试剂来监测体内 GTP 水平和 GTP/GDP 比率

• 批准号: 8129628
• 负责人: RUI J. SOUSA
• 金额: $ 18.8万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8129628
• 关键词: Address; Aging; Binding Proteins; Biological Assay; Biomedical Research; Cell Cycle; Cell physiology; Cells; Cellular Metabolic Process; Development; Disease; Energy Supply; Engineering; Exhibits; Fluorescence; GTP Binding; GTP-Binding Proteins; Guanosine Triphosphate; Health; Human Pathology; Image; Insecta; Life; Light; Luciferases; Malignant Neoplasms; Measures; Metabolism; Methods; Monitor; Nature; Play; Reagent; Resolution; Role; Technology; Variant; Work; anticancer research; human disease; in vivo; insight; luminescence; protein function; protein phosphatase inhibitor-2; public health relevance; sensor; tool

DESCRIPTION (provided by applicant): Cellular processes intimately tied to cancer and to a variety of other human pathologies, are globally regulated or coordinated by GTP and ATP, molecules that represent the energy currency of the cell and which are also the most common allosteric modulators of protein function. Methods to measure the levels of these molecules in vivo, with high temporal and spatial resolution, would be invaluable in understanding how variations in GTP and ATP concentrations regulate and coordinate cellular metabolic processes, and in elucidating the role played by disruption of cellular metabolism in cancer and other diseases. Such methods-luminescence by insect luciferase and fluorescence from GFP- ATP binding protein fusions-exist for ATP. The luciferase assay is widely used and has led to important recent discoveries regarding the role of variations in ATP levels in the cell cycle and human disease, including cancer. However, no equivalent methods exist for GTP, even though GTP, through its action on numerous G-proteins, arguably plays a larger regulatory role in the cell than ATP. To address the need for such technology we will: (1) Engineer insect luciferase so that it will specifically use GTP, rather than ATP, to generate light, and (2) Engineer GFP-G-protein fusions that will exhibit altered excitation spectra upon binding GTP. These genetically encoded sensors will provide two complementary methods for monitoring GTP levels and GTP/GDP ratios inside living cells with high temporal and spatial resolution. PUBLIC HEALTH RELEVANCE: Cancer and many other human diseases, especially those associated with aging, often involve changes in cellular metabolism: alterations in the nature and concentrations of the molecules that supply the energy for cellular activity and dis-coordination in the network of the many cellular processes normally involved in maintaining cellular health. Insight into how metabolic processes are coordinated and regulated in a healthy cell can come from monitoring the levels of regulatory molecules, but this can be challenging to do in living cells. The proposed work will make it possible to monitor levels of GTP, one of the most important of these regulatory molecules, and will therefore create an invaluable tool for cancer and biomedical research.

描述（由申请人提供）：与癌症和多种其他人类病理学密切相关的细胞过程由GTP和ATP进行全局调节或协调，GTP和ATP是代表细胞能量流通的分子，也是蛋白质功能的最常见变构调节剂。以高时间和空间分辨率测量这些分子在体内水平的方法对于理解GTP和ATP浓度的变化如何调节和协调细胞代谢过程以及阐明细胞代谢中断在癌症和其他疾病中所起的作用将是非常宝贵的。这种方法-昆虫荧光素酶发光和GFP- ATP结合蛋白融合的荧光-存在ATP。荧光素酶测定被广泛使用，并导致了关于细胞周期和人类疾病（包括癌症）中ATP水平变化的重要最新发现。然而，没有等价的方法存在GTP，即使GTP，通过其对许多G-蛋白的作用，可以说在细胞中起着比ATP更大的调节作用。为了解决对这种技术的需求，我们将：（1）工程化昆虫荧光素酶，使其特异性地使用GTP而不是ATP来产生光，以及（2）工程化GFP-G-蛋白融合体，其在结合GTP时将表现出改变的激发光谱。这些基因编码的传感器将提供两种互补的方法，用于以高的时间和空间分辨率监测活细胞内的GTP水平和GTP/GDP比率。 公共卫生关系：癌症和许多其他人类疾病，特别是与衰老有关的疾病，通常涉及细胞代谢的变化：为细胞活动提供能量的分子的性质和浓度的改变，以及通常参与维持细胞健康的许多细胞过程网络的不协调。了解代谢过程如何在健康细胞中协调和调节可以来自监测调节分子的水平，但这在活细胞中可能具有挑战性。拟议的工作将使监测这些调节分子中最重要的分子之一的鸟苷三磷酸水平成为可能，因此将为癌症和生物医学研究创造一个宝贵的工具。