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

描述（由申请人提供）：一氧化氮（NO）是一种小的反应性分子，参与多种信号传导途径，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酸环化酶 (sGC)，它是一种 150 kDa 的异二聚体血红蛋白，也是治疗包括癌症在内的疾病的有吸引力的靶标。 NO 的结合刺激 sGC 活性，导致 cGMP 信号级联的建立。 sGC 受到多种分子的变构调节，包括 NO、ATP、YC-1（一种小的核苷酸样药效基团）以及翻译后修饰。尽管进行了广泛的研究，但人们对 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细胞中蛋白质的排列知之甚少。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，我们打算将配对荧光团合并到全长和截短形式的 sGC 中，以便 FRET 测量能够揭示刺激和抑制条件下功能域之间的距离。我们开发了一个强大的模型系统，涉及天蛾（Manduca sexta）的 sGC，以此开始这些研究，但一旦荧光系统建立，也将包括人类 sGC。我们不仅将研究分离材料的 sGC 构象状态，还将研究活细胞中的 sGC 构象状态。此外，我们将结合使用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 在细胞中呈现出点状排列，但这种鲜明模式的功能后果尚不清楚。我们预计这些研究将揭示 sGC 经历的结构转变，并为药物发现的新策略提供框架。该试点项目产生的结果将为未来的合作资助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.