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

描述（由申请人提供）：细胞过程与癌症和各种其他人类病理密切相关，由GTP和ATP全局调节或协调，GTP和ATP是代表细胞能量货币的分子，也是蛋白质功能最常见的变构调节剂。在体内以高时间和空间分辨率测量这些分子水平的方法，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，以及阐明细胞代谢破坏在癌症和其他疾病中所起的作用，将是非常宝贵的。昆虫荧光素酶发光法和GFP- ATP结合蛋白融合荧光法是针对ATP存在的两种发光方法。荧光素酶测定被广泛使用，并导致了关于ATP水平变化在细胞周期和人类疾病（包括癌症）中的作用的重要发现。然而，尽管GTP通过作用于许多g蛋白，可以说在细胞中发挥着比ATP更大的调节作用，但目前还没有针对GTP的等效方法。为了满足对这种技术的需求，我们将：(1)设计昆虫荧光素酶，使其专门使用GTP而不是ATP来产生光；(2)设计gfp - g蛋白融合物，使其在结合GTP时表现出改变的激发光谱。这些基因编码传感器将提供两种互补的方法来监测活细胞内GTP水平和GTP/GDP比率，具有高时空分辨率。

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.