The Partnership for Native American Cancer Prevention (1 of 2)

描述（由申请人提供）：一氧化氮（NO）是一种小的反应性分子，参与许多信号传导途径，包括调节血管生成和转移的信号传导途径。NO的主要细胞受体是可溶性鸟苷酰环化酶（sGC），其是150 kDa的异二聚体血红素蛋白，并且是用于治疗疾病（包括癌症）的有吸引力的靶标。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（包括NO、ATP、YC-1（一种小核苷酸样药效团））和翻译后修饰的变构调节。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、蛋白质中功能结构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，并监测细胞内的sGC定位。具体而言，我们打算将成对的荧光团纳入全长和截短形式的sGC，使得FRET测量将揭示在刺激和抑制条件下功能结构域之间的距离。我们已经开发了一种稳健的模型系统，该系统涉及来自天蛾（Manduca sexta）的sGC，开始这些研究，但是一旦建立荧光系统，还将包括人sGC。我们将研究sGC构象状态不仅与分离的材料，而且在活细胞。此外，我们将使用免疫组织学和荧光显微镜的组合来监测激活和抑制条件下sGC的定位。我们已经证明sGC在细胞中显示出点状排列，但这种明显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。这一试点项目产生的成果将为今后的合作供资和研究工作奠定基础。

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.