Nitric oxide induced soluble guanylate cyclase dysfunction or activation: Implications as disease biomarkers 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：作为疾病生物标志物或在治疗中的意义

• 批准号: 10002614
• 负责人: Arnab Ghosh
• 金额: $ 41.36万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-15 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10002614
• 关键词: A549; Albumins; Apical; Asthma; Biochemical; Biological Markers; Biology; Bronchodilation; Bronchodilator Agents; Cell Communication; Chronic; Coculture Techniques; Disease; Dose; Enzymes; Epigenetic Process; Epithelial Cells; Epithelium; 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; NOS2A gene; Nitric Oxide; Oxidation-Reduction; Pathway interactions; Pharmaceutical Preparations; 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; 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. 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.

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未知，来自 我们的小组发现伴侣热休克蛋白90在血红素插入三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) 和血红蛋白 (Hb)。我们的 研究表明，强烈的 sGC-hsp90 相互作用可以衡量细胞中无血红素的 sGC，并且这 就 sGC 亚基异二聚化而言，相互作用是相互排斥的。总之，这些发现 在 sGC 功能失调疾病的临床诊断中的潜在应用。我们发现 在一氧化氮 (NO) 升高的情况下，炎症性哮喘中 sGC 会出现功能障碍，从而阻碍 基于 NO 的支气管扩张，但可以通过 sGC 激活剂来克服，尽管 sGC 激活剂可以诱导支气管扩张 这个损失。哮喘中的这种 sGC 功能障碍与 sGC 功能障碍的强烈分子特征相关 其中包含弱 sGC-α1β1 异二聚体、强 sGCβ1-hsp90 相互作用和高 S-亚硝基化 (SNO) 于 sGC-β1。我们当前和过去的研究表明，NO 水平在生物学中至关重要，并且可以发挥作用 sGC 的成败都有两种方式。虽然哮喘中的高 NO 水平可以通过破坏 sGC-α1β1异二聚体，低NO水平可以触发sGC-β1中的血红素插入，增加和稳定 sGC异二聚体。此外，在人类哮喘 ASMC（气道平滑肌细胞）中，我们的研究表明 sGC 由于血红素缺乏而功能失调，但可以被 sGC 激活剂激活。基于这些 令人兴奋的新发现，我们建议 (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。这包括确定脱亚硝基酶是否存在的机制 如硫氧还蛋白-1（Trx-1）或NO清除剂Hb在根尖上皮表达可起到保护作用 用于底层气道平滑肌 sGC。 (ii) 建立两种 sGC 功能障碍的分子标志 小鼠哮喘模型（OVA 白蛋白和屋尘螨模型 [HDME]）和人类严重哮喘模型 HASMC 和肺组织样本。 (iii) 确定引起的遗传、表观遗传和生化机制 有缺陷的 sGC。 (iv) 探索恢复严重哮喘HASMC sGC功能的方法，包括治疗 NO 暴露和过度表达有益蛋白（Hsp90、Trx-1、过氧化氢酶），其表达可能是 哮喘 HASMC 中降低。我们的项目将共同推进目前关于伴侣如何、 氧化还原酶、NO 和炎症调节健康气道和哮喘气道中的 sGC，并提出了以下方法： 恢复其功能。