The Partnership for Native American Cancer Prevention (2 of 2)

描述（由申请人提供）：一氧化氮（NO）是一种小的活性分子，参与许多信号通路，包括调节血管生成和转移。NO的主要细胞受体是可溶性冠酰环化酶（sGC），这是一种150 kDa的异二聚体血红蛋白，是治疗包括癌症在内的疾病的一个有吸引力的靶点。NO的结合刺激sGC活性，导致cGMP信号级联的建立。sGC受多种分子的变构调节，包括NO、ATP、YC-1（一种小核苷酸样药效团）和翻译后修饰。尽管进行了广泛的研究，但对sGC的整体形状、变构调节发生的方式、蛋白质中功能域的排列或蛋白质在细胞中的排列知之甚少。我们打算通过基于荧光的方法来填补这一空白，这将使我们能够测量sGC内的结构变化，并监测细胞内sGC的定位。具体来说，我们打算将成对的荧光团结合到全长和截断形式的sGC中，这样FRET测量将揭示在刺激和抑制条件下功能域之间的距离。我们已经开发了一个强大的模型系统，其中包括来自鹰蛾（Manduca sexta）的sGC，作为这些研究的开始，但一旦荧光系统建立起来，也将包括人类sGC。我们将不仅在分离材料中研究sGC的构象状态，而且在活细胞中研究sGC的构象状态。此外，我们将结合免疫组织学和荧光显微镜来监测激活和抑制条件下sGC的定位。我们已经证明，sGC在细胞中显示出标点排列，但这种鲜明模式的功能后果尚不清楚。总之，我们期望这些研究将揭示sGC经历的结构转变，并为药物发现的新策略提供框架。这个试点项目产生的结果将为今后的合作资助和研究工作提供基础。

DESCRIPTION (provided by applicant): Nitric oxide (NO) is a small, reactive molecule involved in numerous signaling pathways, including those regulating angiogenesis and metastasis. The primary cellular receptor for NO is soluble Guanylyl Cyclase (sGC), a heterodimeric hemoprotein of 150 kDa and an attractive target for the treatment of disease, including cancer. Binding of NO stimulates sGC activity, leading to the establishment of a cGMP signaling cascade. sGC is allosterically regulated by a variety of molecules, including NO, ATP, YC-1 (a small nucleotide-like pharmacophore), and by posttranslational modifications. Despite extensive study, little is known about the overall shape of sGC, the means by which allosteric regulation takes place, the arrangement of functional domains in the protein or the arrangement of the protein in the cell. We intend to fill this gap through fluorescence-based approaches that will allow us to measure structural changes within sGC, and also to monitor sGC localization within the cell. Specifically, we intend to incorporate paired fluorophores to full-length and truncated forms of sGC such that FRET measurements will reveal the distances between functional domains under stimulating and inhibiting conditions. We have developed a robust model system involving sGC from the hawk moth {Manduca sexta) with which to begin these studies, but will also include human sGC once the fluorescence system is established. We will investigate sGC conformational states not only with isolated material, but also in live cells. Additionally, we will use a combination of immunohistology and fluorescence microscopy to monitor localization of sGC under activating and inhibiting conditions. We have shown that sGC displays a punctuate arrangement in the cell, but the functional consequences of this stark pattern are unknown. Together, we expect that these studies will uncover the structural transitions that sGC undergoes and provide the framework for novel strategies in drug discovery. The generated results from this pilot project will provide the basis for future collaborative funding and research efforts.