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

一氧化氮诱导可溶性鸟苷酸环化酶功能障碍或激活：作为疾病指标或治疗的意义

• 批准号: 10217246
• 负责人: Arnab Ghosh
• 金额: $ 40.36万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-15 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10217246
• 关键词: A549; Apical; Asthma; Biochemical; Biology; Blood Platelets; Bronchodilation; Bronchodilator Agents; Cell Communication; Cells; Chronic; Coculture Techniques; Coupling; Disease; Dose; Enzymes; Epigenetic Process; Epithelial; Epithelial Cells; Freund' s Adjuvant; Functional disorder; Future; Genetic; Heat-Shock Proteins 90; Heme; Hemeproteins; Hemoglobin; Heterodimerization; Human; Impairment; Inflammation; Inflammatory; Knowledge; Life; Lung; Mammals; Measures; Modeling; Molecular; Molecular Chaperones; Molecular Profiling; Mus; Muscle relaxation phase; Myoglobin; NOS2A gene; Nitric Oxide; Ovalbumin; Pathway interactions; Pharmaceutical Preparations; Phenotype; Production; Proteins; Pyroglyphidae; Research; Role; SKIL gene; Sampling; Signal Transduction; Slice; Smooth Muscle; Smooth Muscle Myocytes; Soluble Guanylate Cyclase; Structure of parenchyma of lung; TXN gene; Testing; Therapeutic; Tissue Sample; Tissues; Work; asthma model; asthmatic; asthmatic airway; asthmatic airway smooth muscle; asthmatic patient; base; catalase; clinical Diagnosis; disease diagnosis; in vivo; insight; mouse model; overexpression; preclinical study; respiratory smooth muscle; response

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.

抽象的 血蛋白对生命至关重要，血红素插入是其成熟和功能的重要步骤。 尽管哺乳动物在成熟期间哺乳动物插入血红素的机制大多未知，但 我们小组的研究发现了伴侣Hsp90在血红素插入四个键中的特定参与 血蛋白，诱导型一氧化氮合酶（iNOS），可溶性鸟叶基环酶（SGC），血红蛋白（Hb）和 肌红蛋白（MB）。我们的研究表明，强烈的SGC-HSP90相互作用可以是无血红素SGC的量度 在细胞中，相对于SGC-Subunit异二聚化，这种相互作用是互斥的。一起， 这些发现在SGC为的临床诊断中具有潜在的应用 功能失调。我们发现SGC在升高的一硝酸下在炎症性哮喘中功能失调 氧化物（否），阻碍了无基基支气管扩张，但可以通过SGC激活剂克服 诱导支气管扩张目的地此损失。哮喘中这种SGC功能障碍与强分子有关 SGC功能障碍的签名包括弱SGC-α1β1异二聚体，强大的SGCβ1-HSP90 SGC-β1上的相互作用和高硝基化（SNO）。我们目前和过去的研究表明没有 水平对生物学至关重要，可以采取两种制造或破坏SGC的方式。虽然没有像哮喘一样高的水平 可以通过破坏SGC-α1β1异二聚体诱导SGC功能障碍，低水平可以触发血红素插入 在SGC-β1中，增加和稳定SGC异二聚体。此外，在人类哮喘ASMC中（气道 平滑肌细胞），我们的研究表明，SGC没有反应，因为它是血红素缺乏的，但是 可以被SGC激活剂激活。根据这些令人兴奋的新发现，我们提出了（i）分子和细胞 - 定义机制的水平研究，以了解SGC在哮喘中如何在高NO下变得功能失调。 这包括确定反硝基糖基酶（例如硫氧还蛋白-1（TRX-1））或HB中存在的机制 顶上上皮（作为A549细胞在我们的新发现中表达HB）可以在基础气道上具有保护作用 平滑肌SGC。 （ii）确定哮喘性HASMC中是否存在有缺陷的SGC血红素（人类气道 平滑肌细胞）或在哮喘（OVA，CFA/HDME）的小鼠模型中导致支气管扩张缺陷 探索该血红素缺陷型SGC的基础，并首先在SGC功能障碍中建立分子特征 人类哮喘，以后可以将其作为活血小板中SGC的功能障碍指标应用 哮喘患者。 （iii）最终将低NO水平在诱导的SGC异二聚化和 在哮喘HASMC中下调的过表达酶（HSP90，TRX-1，过氧化氢酶），我们建议 在此类HASMC中恢复SGC功能障碍，该HASMC显示了Predomian无血红素SGC表型。在一起 项目将促进当前有关HSP90，NO和炎症如何调节SGC成熟的知识， 并将提供有关健康和哮喘气道中SGC成熟的新信息。