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: 10657664
• 负责人: Arnab Ghosh
• 金额: $ 40.19万
• 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 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10657664
• 关键词: A549; Apical; Asthma; Biochemical; Biology; Blood Platelets; Bronchodilation; Bronchodilator Agents; Cells; Chronic; Coculture Techniques; Coupling; Disease; Dose; Enzymes; Epigenetic Process; Epithelial Cells; Epithelium; 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; Ovum; Pathway interactions; Pharmaceutical Preparations; Phenotype; Production; Proteins; Pyroglyphidae; Research; Role; 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; catalase; clinical diagnosis; disease diagnosis; functional restoration; 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 血红素蛋白对于生命至关重要，血红素插入是其成熟和功能的重要步骤。 尽管哺乳动物在血红素蛋白成熟过程中插入血红素的机制大多未知， 我们小组的研究揭示了伴侣 hsp90 在血红素插入四个关键中的具体参与 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 我们的研究表明，强 sGC-hsp90 相互作用可以衡量无血红素 sGC。 在细胞中，这种相互作用与 sGC 亚基异二聚化是相互排斥的。 这些发现在 sGC 疾病的临床诊断中具有潜在的应用 我们发现，在硝酸盐升高的情况下，炎症性哮喘中 sGC 会出现功能障碍。 氧化物 (NO)，它会阻碍基于 NO 的支气管扩张，但可以通过 sGC 激活剂来克服，sGC 激活剂可以 尽管存在这种损失，但仍会诱导支气管扩张。哮喘中的这种 sGC 功能障碍与强分子相关。 sGC 功能障碍的特征，包括弱的 sGC-α1β1 异二聚体、强的 sGCβ1-hsp90 sGC-β1 上的相互作用和高 S-亚硝基化 (SNO) 我们当前和过去的研究表明，NO。 NO 水平在生物学中至关重要，并且可以通过两种方式发挥作用来形成或破坏 sGC，而高 NO 水平则如哮喘。 可以通过破坏 sGC-α1β1 异二聚体来诱导 sGC 功能障碍，低 NO 水平可以触发血红素插入 在 sGC-β1 中，增加并稳定 sGC 异二聚体。 平滑肌细胞），我们的研究表明 sGC 由于血红素缺乏而对 NO 没有反应，但是 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- 水平研究来定义机制，以了解 sGC 在高 NO 条件下（如哮喘）如何变得功能障碍。 这包括确定脱亚硝基酶（例如硫氧还蛋白-1 (Trx-1) 或 Hb）是否存在于 心尖上皮（在我们的新发现中，A549 细胞表达 Hb）可以对下层气道发挥保护作用 (ii) 确定哮喘 HASMC（人气道）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）导致支气管扩张缺陷， 探索这种血红素缺陷 sGC 的基础，并牢固地建立 sGC 功能障碍的分子特征 人类哮喘，因此这可以在未来应用作为活体血小板中 sGC 功能障碍的指标 (iii)最后结合低NO水平诱导sGC异二聚化的作用，以及 哮喘 HASMC 中过度表达下调的酶（Hsp90、Trx-1、过氧化氢酶），我们建议 恢复显示主要无血红素 sGC 表型的 HASMC 中的 sGC 功能障碍。 该项目将推进目前关于 hsp90、NO 和炎症如何调节 sGC 成熟的知识， 并将提供有关健康气道和哮喘气道中 sGC 成熟的新信息。