Nitric Oxide Signaling in Health and Disease

 描述(申请人提供)：这是一项了解一氧化氮受体--可溶性鸟苷酸环化酶(SGC)的结构、功能、调节和小分子刺激的新方案。一氧化氮(NO)值得注意的是，它是一种小而有毒的化合物，但由大多数细胞产生，以调节不同的生理，包括血压、伤口愈合、组织发育和记忆形成。NO信号的缺失会导致心血管疾病，并与糖尿病、衰老和相关的健康衰竭相关的血管疾病有关。有希望的靶向sGC的新化合物正在临床试验或临床中，但它们的功能如何，甚至在许多情况下它们在哪里结合尚不清楚，这使得进展具有挑战性。SGC是一种150 kDa的异二聚体血红蛋白，它通过变构调节对多种因素做出反应，包括NO和候选药物的刺激。我们已经开发了人和鹰蛾(Manduca Sexta)sGC，用于解决NO信号的机制。我们试图在分子水平上了解sGC在正常生理中是如何发挥作用的，它在心血管疾病中是如何失败的，以及药物结合如何克服这些失败。我们提出了三个具体目标：目标1：sGC及其功能络合物的原子结构。一氧化氮领域的一个关键限制是缺乏一氧化氮受体的结构。在存在或不存在NO和刺激剂化合物的情况下，我们合成了截断的异二聚体sGC的新晶体。这些晶体应该允许确定活性和非活性状态的原子结构，为sGC激活提供关键的见解，并揭示药物结合和药物作用的分子细节。目的2：sGC的信号转导机制。NO或候选药物与sGC的结合导致催化活性增加约200倍，但如何从血红素结构域到环化酶结构域的信号转导尚不清楚。我们建议通过紫外可见光谱和发光衰减光谱测量、工程稀土结合标签和诱变来揭示sGC激活的潜在信号转导机制。目的3：通过翻译后修饰对SGC进行调控。SGC对NO的反应是以多种方式调节的，但最重要的是通过磷酸化。我们建议发现sGC在哪里被磷酸化以响应细胞外信号，磷酸化是如何抑制蛋白质的，哪条信号通路是负责的，以及它何时发生在细胞内。采用的主要技术是质谱学。我们先前已经证明，YC-1家族候选药物可以通过磷酸化来克服sGC抑制。我们预计，对细胞sGC调控的更全面了解将提供亟需的药物发现机会。

 DESCRIPTION (provided by applicant): This is a new proposal to understand the structure, function, regulation and small-molecule stimulation of the nitric oxide receptor, soluble guanylyl/guanylate cyclase (sGC). Nitric oxide (NO) is remarkable in that it is a small, toxic compound, but is produced by most cells to regulate diverse physiology, including blood pressure, wound healing, tissue development and memory formation. Loss of NO signaling leads to cardiovascular disease and is linked to the vascular disorders associated with diabetes, aging and related health failures. Promising new compounds targeting sGC are in clinical trial or in the clinic but how they function or even where they bind in many cases is unknown, making advancement challenging. sGC is a 150 kDa heterodimeric hemoprotein that responds to multiple factors through allosteric regulation, including stimulation by NO and by drug candidates. We have developed human and hawkmoth (Manduca sexta) sGC for addressing mechanism in NO signaling. We seek to understand, at a molecular level, how sGC functions in normal physiology, how it fails in cardiovascular disease and how drug binding can overcome these failures. We propose three specific aims: Aim 1: Atomic structures of sGC and its functional complexes. A key limitation in the nitric oxide field is the absence of structure for th nitric oxide receptor. We have produced new crystals of truncated heterodimeric sGC in the presence or absence of NO and stimulator compounds. These crystals should allow for atomic structures to be determined for both the active and inactive states, providing key insight into sGC activation and revealing the molecular details for drug binding and drug action. Aim 2: Mechanism for signal transduction in sGC. Binding of NO or candidate drugs to sGC leads to an ~200 fold increase in catalytic activity, but how signal transduction from the heme domain to the cyclase domain takes place is unknown. We propose to uncover the underlying signal transduction mechanism for sGC activation using UV-visible and luminescence decay spectroscopic measurements, engineered lanthanide binding tags and mutagenesis. Aim 3: sGC regulation through posttranslational modification. sGC response to NO is regulated in multiple ways, but most importantly through phosphorylation. We propose to discover where sGC is phosphorylated in response to extracellular signals, how phosphorylation inhibits the protein, which signaling pathway is responsible and when it occurs in the cell. The primary technique to be employed is mass spectrometry. We have previously shown that YC-1 family drug candidates can overcome sGC inhibition by phosphorylation. We expect a more complete understanding of cellular sGC regulation will provide much needed drug-discovery opportunities.