Regulation of Soluble Guanylyl Cyclase, the NO-Receptor

摘要 一氧化氮（NO）和细胞氧化还原信号传导是重要参与生理学和细胞凋亡的相关途径。 心血管系统的病理生理学。NO的主要受体是可溶性鸟苷酸环化酶（GC 1）， 含血红素异二聚体。当NO与血红素结合时，GC活性被刺激数百倍 生产cGMP。尽管NO-cGMP通路在血管内稳态和血管内皮细胞的生长中起着关键作用， 尽管GC 1在病理生理学上是重要的，但GC 1的调节和激活机制仍然知之甚少。 我们先前表明，GC 1的特定Cys的S-亚硝化和其他硫醇氧化导致其成为 对NO催化刺激脱敏，这是一种具有治疗重要性的现象。事实上，GC 1是 最受欢迎的治疗心血管疾病的靶点，特别是克服NO抗性， 血管功能障碍（即，当外源性NO不能纠正被破坏的血管反应性时）。 我们发现GC 1通过混合二硫键交换与硫氧还蛋白1（Trx 1）相互作用，这种相互作用 似乎保护GC 1免受对NO刺激的脱敏。有趣的是，GC 1-Trx 1复合物 通过用血管紧张素II和S-亚硝基半胱氨酸处理诱导细胞巯基氧化而增加。我们最 最近的研究揭示了GC 1中存在二硫键，并表明这些二硫键 未刺激（基础）和NO刺激条件之间的差异，表明巯基/二硫键开关 可能参与了GC 1的激活机制。 基于新的生物化学、蛋白质组学和结构证据，我们提出GC 1从 基础水平的催化（在没有NO的情况下产生cGMP）到高速率的cGMP产生， 对NO-血红素结合的反应是通过特异性二硫键的断裂和潜在的 不同的二硫键以产生和稳定高活性催化构象。此外，我们将探讨 Trx 1和GC 1之间的相互作用参与了 通过促进高催化状态的二硫化物的还原以促进返回 到基础状态和敏化一个新的循环的NO激活。利用细胞，生化， 分子动力学模拟和质谱实验，我们将识别和确定 Aim 1中二硫键的功能，并建立GC 1相互作用的机制和生物学相关性 目标2中的Trx 1。 该项目背后的统一思想是心血管生物学中的NO信号通过经典的 NO-cGMP途径依赖于GC 1的反应性二硫化物、它们的氧化还原调节和它们的氧化还原- 与其他蛋白质的相互作用。心血管功能和功能障碍的病因可能非常 很好地依赖于保持GC 1硫醇/二硫键开关的适当平衡的能力。

ABSTRACT Nitric oxide (NO) and cellular redox signaling are linked pathways crucially involved in the physiology and pathophysiology of the cardiovascular system. The main receptor for NO is soluble guanylyl cyclase (GC1), a heme-containing heterodimer. Upon binding of NO to the heme, GC activity is stimulated several hundred-fold to produce cGMP. Despite the critical role of the NO-cGMP pathway in vascular homeostasis and pathophysiology, the mechanisms of regulation and activation of GC1 are still poorly understood. We previously showed that S-nitrosation and other thiol oxidations of specific Cys of GC1 causes it to become desensitized to NO catalytic stimulation, a phenomenon of therapeutic importance. In fact, GC1 is one of the most sought-after targets for treatment of cardiovascular diseases, in particular to overcome NO resistance in vascular dysfunction (i.e., when exogenous NO cannot correct disrupted vascular reactivity). We discovered that GC1 interacts with thioredoxin 1 (Trx1) via a mixed disulfide exchange and this interaction appears to protect GC1 from desensitization to NO stimulation. Interestingly, the GC1-Trx1 complex was increased by inducing cellular thiol oxidation with Angiotensin II and S-nitrosocysteine treatments. Our most recent investigations reveal the presence of disulfide bonds in GC1 and indicate that these disulfide bonds are different between unstimulated (basal) and NO-stimulated conditions, suggesting that thiol/disulfide switches could be involved in the mechanism of activation of GC1. Based on novel biochemical, proteomic and structural evidence, we propose that the transition of GC1 from basal levels of catalysis (generation of cGMP in the absence of NO) to high rates of cGMP production in response to NO-heme binding is mediated by breaking of specific disulfide bonds and potential formation of different disulfide bonds to create and stabilize a highly active catalytic conformation. Moreover, we will explore the hypothesis that the interaction between Trx1 and GC1 is involved in the mechanism of activation/deactivation by facilitating the reduction of disulfide(s) of the high catalytic state to promote the return to basal state and sensitization to a new cycle of NO activation. Using a combination of cellular, biochemical, Molecular Dynamics simulation and Mass Spectrometry experiments, we will identify and determine the function of disulfide bonds in Aim1 and establish the mechanism and biological relevance of GC1 interaction with Trx1 in Aim2. The unifying idea behind this project is the concept that NO signaling in cardiovascular biology via the classical NO-cGMP pathway is dependent on reactive disulfide(s) of GC1, their redox modulation and their redox- dependent interaction with other proteins. The etiology of cardiovascular function and misfunction could very well depend on the ability to maintain proper balance of GC1 thiol/disulfide switches.