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

描述(申请人提供)：与癌症和其他各种人类病理密切相关的细胞过程，由GTP和ATP全球调节或协调，这两个分子代表细胞的能量流通，也是蛋白质功能最常见的变构调节器。在体内以高时间和空间分辨率测量这些分子的水平，对于了解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，将是非常有价值的。这样的方法--昆虫荧光素酶发光和GFP-ATP结合蛋白融合的荧光--存在于ATP中。荧光素酶分析被广泛使用，并导致了最近关于ATP水平变化在细胞周期和包括癌症在内的人类疾病中的作用的重要发现。然而，尽管GTP通过对大量G-蛋白的作用，在细胞中发挥着比ATP更大的调节作用，但目前还没有类似的方法来研究GTP。为了满足这种技术的需求，我们将：(1)改造昆虫荧光素酶，使其专门使用GTP而不是ATP来发光，以及(2)改造GFP-G-蛋白质融合，使其在结合GTP时表现出不同的激发光谱。这些基因编码的传感器将提供两种互补的方法，以高时间和空间分辨率监测活细胞内的GTP水平和GTP/GDP比率。 公共卫生相关性：癌症和许多其他人类疾病，特别是与衰老相关的疾病，往往涉及细胞新陈代谢的变化：为细胞活动提供能量的分子的性质和浓度的变化，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。深入了解健康细胞中的代谢过程是如何协调和调节的，可以通过监测调节分子的水平来实现，但在活细胞中做到这一点可能是具有挑战性的。这项拟议的工作将使监测GTP水平成为可能，GTP是这些最重要的调节分子之一，因此将为癌症和生物医学研究创造一个宝贵的工具。

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.