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

摘要 血红素蛋白是生命所必需的，而插入血红素是其成熟和功能的关键步骤。 虽然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尚不清楚， 我们小组的研究发现，伴侣HSP90参与了将血红素插入四个键的特定过程 血红蛋白、诱导型一氧化氮合酶(INOS)、可溶性鸟苷酸环化酶(SGC)、血红蛋白(Hb)和 肌红蛋白(Mb)。我们的研究表明，sGC-HSP90的强相互作用可以作为无血红素sGC的一种衡量标准 这种相互作用与sGC-亚单位异源二聚化是互斥的。一起， 这些发现在sGC所在疾病的临床诊断中具有潜在的应用价值。 功能失调。我们发现，在炎症性哮喘中，sGC在一氧化氮升高的情况下会出现功能障碍 氧化物(NO)，它阻碍了基于NO的支气管扩张，但可以被sGC激活剂克服，sGC激活剂可以 尽管有这样的损失，还是要诱导支气管扩张。哮喘的这种sGC功能障碍与一种强烈的分子相关 SGC功能障碍的特征，包括弱的sGC-α1β1异二聚体，强的sGCβ1-hsp90 在sGC-β1上的相互作用和高的SNO。 水平在生物学上是至关重要的，可以通过两种方式决定sGC的成败。虽然哮喘患者体内的NO水平较高 可通过破坏sGC-α1β1异二聚体而导致sGC功能障碍，低水平的NO可触发血红素插入 在sGC-β1中，增加和稳定sGC杂二聚体。此外，在人类哮喘的ASMCs(呼吸道 我们的研究表明，sGC对NO没有反应，因为它缺乏血红素，但是 可以被sGC激活剂激活。基于这些令人振奋的新发现，我们提出(I)分子和细胞- 水平研究，以确定机制，以了解sGC如何在高NO下变得功能失调，如在哮喘中。 这包括确定脱氮酶，如硫氧还蛋白-1(TRX-1)或Hb是否存在于 顶端上皮(A549细胞在我们的新发现中表达Hb)可以对下呼吸道起到保护作用。 平滑肌SGC。(2)确定哮喘HASMC(人的呼吸道)中是否存在有缺陷的sGC血红素 平滑肌细胞)或哮喘小鼠模型(OVA、CFA/HDME)导致有缺陷的支气管扩张， 探讨这种血红素缺乏的sGC的基础，并牢固地建立sGC功能障碍的分子特征。 人类哮喘，使其在未来可作为活体血小板sGC功能障碍的指标应用 哮喘患者。(3)低水平NO诱导sGC异二聚化的偶联作用； 在哮喘HASMC中过表达下调的酶(Hsp90，TRX-1，CAT)，我们建议 恢复这类HASMC的sGC功能障碍，这些细胞表现出主要的无血红素sGC表型。在一起我们的 该项目将推进关于HSP90、NO和炎症如何调节sGC成熟的现有知识， 并将为sGC在健康和哮喘呼吸道中的成熟提供新的信息。

ABSTRACT Hemeproteins are essential for life, and heme insertion is an essential step in their maturation and function. 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 four key hemeproteins, inducible nitric oxide synthase (iNOS), soluble guanylyl cyclase (sGC), hemoglobin (Hb) and myoglobin (Mb). 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 of 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 unresponsive to NO due to it being heme deficient, but can be activated by sGC activators. Based on these exciting new findings we propose (i) molecular and cellular- level studies to define mechanisms to understand how sGC becomes dysfunctional under high NO as in asthma. This includes mechanisms to determine whether a denitrosylase such as thioredoxin-1 (Trx-1) or Hb present in the apical epithelium (as A549 cells express Hb in our new find) can have a protective role for underlying airway smooth muscle sGC. (ii) Establish, whether a defective sGC heme exists in asthmatic HASMCs (human airway smooth muscle cells) or in mouse models of asthma (OVA, CFA/HDME) causing defective bronchodilation, explore the basis of this heme-deficient sGC and firmly establish the molecular signatures of sGC dysfunction in human asthma, such that this can be applied in future as a dysfunction indicator of sGC in blood platelets of live asthma patients. (iii) Finally coupling the effect of low NO levels in inducing sGC heterodimerization, and overexpressing enzymes which are downregulated (Hsp90, Trx-1, Catalase) in asthmatic HASMCs, we propose to restore sGC dysfunction in such HASMCs that display a predomiant heme-free sGC phenotype. Together our project will advance the current knowledge of how hsp90, NO and inflammation can regulate sGC maturation, and will provide new information on sGC maturation in healthy and asthmatic airways.