Nitric oxide induced soluble guanylate cyclase dysfunction or activation: Implications as disease biomarkers or in therapy

摘要 血红素蛋白是生命所必需的，而插入血红素是它们成熟过程中必不可少的一步。尽管 哺乳动物在血红素蛋白成熟过程中插入血红素的机制大多未知，研究来自 我们的团队发现了伴侣HSP90在将血红素插入到三个键中的特定参与 血红蛋白、诱导型一氧化氮合酶(INOS)、可溶性鸟苷酸环化酶(SGC)和血红蛋白(Hb)。我们的 研究表明，sGC-HSP90的强烈相互作用可以作为细胞中不含血红素的sGC的衡量标准，而且这一点 相互作用对于sGC-亚基异源二聚是互斥的。总而言之，这些发现有 在sGC功能障碍的疾病诊断中的潜在应用。我们发现 在炎症性哮喘中，sGC在一氧化氮(NO)升高的情况下变得功能失调，从而阻碍 NO为基础的支气管扩张，但可被sGC激活剂克服，后者可诱导支气管扩张，尽管 这场失利。哮喘患者的sGC功能障碍与sGC功能障碍的强烈分子特征有关 它由弱的sGC-α1β1杂二聚体、强的sGCβ1-热休克蛋白90相互作用和高的S亚硝化组成 (sGC-β1)。我们目前和过去的研究表明，NO水平在生物学上是关键的，可以起作用 这两种方式都能使sgc成败。而哮喘时的高NO水平可通过破坏 SGC-α1β1异源二聚体，低水平的NO可以触发血红素插入sGC-β1，增加和稳定 SGC杂二聚体。此外，我们的研究表明，在人类哮喘的ASMC(呼吸道平滑肌细胞)中， 由于缺乏血红素，SGC功能障碍，但可被SGC激活剂激活。基于这些 我们提出了令人振奋的新发现：(I)确定哮喘sGC功能障碍的分子基础，以及 损害或保护sGC的细胞机制。这包括确定一种脱氮酶是否 如硫氧还蛋白-1(Trx-1)或在根尖上皮中表达的无清除剂Hb可起到保护作用 用于下呼吸道平滑肌SGC。(Ii)确定两个sGC功能障碍的分子特征 小鼠哮喘模型(OVA白蛋白和屋尘螨模型[HDME])和人类重症哮喘 HASMC和肺组织样本。确定遗传、表观遗传和生化机制导致 有缺陷的SGC。(4)探索恢复重症哮喘患者sGC功能的方法，包括治疗 无暴露和过表达有益蛋白(Hsp90、Trx-1、过氧化氢酶)，其表达可能是 哮喘时HASMC降低。我们的项目将共同推进目前对监护人如何， 氧化还原酶、一氧化氮和炎症调节健康和哮喘呼吸道中的sGC，并提出了一些方法 恢复其功能。

ABSTRACT Hemeproteins are essential for life and heme insertion is an essential step in their maturation. Although the mechanisms by which mammals insert heme during hemeprotein maturation are mostly unknown, studies from our group uncovered a specific involvement of the chaperon hsp90 in heme insertion into three key hemeproteins, inducible nitric oxide synthase (iNOS), soluble guanylyl cyclase (sGC) and hemoglobin (Hb). Our studies indicate that a strong sGC-hsp90 interaction can be a measure of heme-free sGC in cells and that this interaction is mutually exclusive with respect to sGC-subunit heterodimerization. Together, these findings have potential applications in the clinical diagnosis of diseased conditions where sGC is dysfunctional. We discovered that sGC becomes dysfunctional in inflammatory asthma under elevated nitric oxide (NO), which impedes the NO-based bronchodilation, but can be overcome by sGC activators which can induce bronchodilation despite this loss. Such sGC dysfunction in asthma is associated with a strong molecular signature of sGC dysfunction which comprises a weak sGC-α1β1 heterodimer, a strong sGCβ1-hsp90 interaction, and a high S-nitrosylation (SNO) on sGC-β1. Our current and past studies have revealed that NO levels are critical in biology and can act both ways to make or break sGC. While high NO levels as in asthma can induce sGC dysfunction by breaking the sGC-α1β1 heterodimer, low NO levels can trigger heme insertion in sGC-β1, increasing and stabilizing the sGC heterodimer. Moreover in human asthmatic ASMCs (airway smooth muscle cells), our studies suggest that sGC is dysfunctional due to it being heme deficient, but can be activated by sGC activators. Based on these exciting new findings we propose (i) to determine the molecular basis of sGC dysfunction in asthma, and the cellular mechanisms that impair or protect sGC. This includes mechanisms to determine whether a denitrosylase such as thioredoxin-1 (Trx-1) or NO scavenger Hb expressed in the apical epithelium can have a protective role for underlying airway smooth muscle sGC. (ii) Establish the molecular hallmarks of sGC dysfunction in two mouse asthma models (OVA albumin and house dust mite model [HDME]) and in human severe asthmatic HASMCs & lung tissue samples. (iii) Determine the genetic, epigenetic, and biochemical mechanisms causing the defective sGC. (iv) Explore means to restore sGC function in severe asthmatic HASMC, including therapeutic NO exposure and overexpressing beneficial proteins (Hsp90, Trx-1, Catalase) whose expression may be lowered in asthmatic HASMCs. Together our project will advance the current knowledge of how chaperones, redox enzymes, NO, and inflammation regulate sGC in healthy and asthmatic airways, and suggest ways to restore its function.