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，血红素插入是其成熟和功能的重要步骤。 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是未知的， 我们小组的研究揭示了伴侣hsp 90在血红素插入四个关键位点中的特异性参与。 血红素蛋白、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，强烈的sGC-hsp 90相互作用可以是无血红素sGC的量度。 并且这种相互作用相对于sGC亚基异源二聚化是互斥的。在一起， 这些发现在临床诊断sGC感染的疾病中具有潜在的应用价值。 功能失调我们发现sGC在炎症性哮喘中在高浓度的硝酸盐下变得功能失调， 氧化物（NO），其阻碍基于NO的支气管扩张，但可以被sGC激活剂克服， 引起支气管扩张。哮喘中的这种sGC功能障碍与一种强分子生物学效应相关。 sGC功能障碍的特征，包括弱sGC-α1β1异二聚体、强sGCβ1-hsp 90 相互作用和sGC-β1上的高S-亚硝基化（SNO）。我们目前和过去的研究表明，没有 水平在生物学中是至关重要的，并且可以以两种方式起作用以形成或破坏sGC。而哮喘患者体内的NO水平 可通过破坏sGC-α1β1异二聚体诱导sGC功能障碍，低NO水平可触发血红素插入 在sGC-β1中，增加并稳定sGC异源二聚体。此外，在人哮喘ASMCs（气道 平滑肌细胞），我们的研究表明sGC对NO无反应，因为它缺乏血红素，但 可以被sGC激活剂激活。基于这些令人兴奋的新发现，我们提出（i）分子和细胞- 水平的研究，以确定机制，以了解如何sGC成为功能失调下高NO哮喘。 这包括确定脱硝酶如硫氧还蛋白-1（Trx-1）或Hb是否存在于 顶端上皮（如我们新发现表达Hb的A549细胞）对下面的气道具有保护作用 平滑肌sGC。(ii)确定哮喘HASMC（人气道）中是否存在缺陷sGC血红素 平滑肌细胞）或哮喘小鼠模型（OVA，CFA/HDME）中引起有缺陷的支气管扩张， 探索这种血红素缺乏的sGC的基础，并牢固地建立sGC功能障碍的分子特征， 人哮喘，使得其可在未来用作肝血小板中sGC功能障碍指示物 哮喘患者(iii)最后，结合低NO水平在诱导sGC异源二聚化中的作用， 在哮喘HASMCs中过度表达下调的酶（Hsp 90，Trx-1，过氧化氢酶），我们提出， 以恢复显示出占优势的无血红素sGC表型的HASMC中的sGC功能障碍。我们一起 该项目将推进目前对hsp 90、NO和炎症如何调节sGC成熟的认识， 并将提供关于健康和哮喘气道中sGC成熟的新信息。

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.