Allostery and Cellular Distribution of the Nitric Oxide Recepto, Soluble...

摘要： 一氧化氮(NO)是一种小的反应性分子，参与了许多信号通路，包括 调节血管生成和转移。一氧化氮的主要细胞受体是可溶性鸟苷酸环化酶 (SGC)，一种150 kDa的异二聚体血红蛋白，是治疗疾病的有吸引力的靶点， 包括癌症。NO结合刺激sGC活性，导致cGMP信号的建立 卡斯卡德。SGC受多种分子的变构调节，包括NO、ATP、YC-1(一种小分子 类似核苷酸的药效团)，以及通过翻译后修饰。尽管进行了广泛的研究，但几乎没有 已知sGC的整体形状，即发生变构调节的方式， 蛋白质中功能区域的排列或蛋白质在细胞中的排列。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，也用于监测SGC在细胞内的定位。具体地说，我们打算将配对 荧光基团到全长和截短形式的sGC，以便FRET测量将揭示 刺激和抑制条件下功能结构域之间的距离。我们已经开发出一种 包括来自鹰蛾(Manduca Sexta)的sGC的稳健模型系统， 但一旦建立了荧光系统，也将包括人类sGC。我们将调查sgc 构象状态不仅存在于分离的物质中，而且存在于活细胞中。此外，我们还将使用 免疫组织学与荧光显微镜结合监测激活状态下sGC的定位 和抑制条件。我们已经展示了sGC在单元格中以点状排列显示，但是 这种严酷模式的功能后果尚不得而知。我们预计，这些研究将共同推动 揭示sGC经历的结构转变并为药物的新策略提供框架 发现号。这一试点项目所产生的结果将为未来的合作筹资提供基础 和研究努力。

ABSTRACT: 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 hawkmoth {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 punctate 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.