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.

摘要 血红素蛋白是生命所必需的，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是未知的， 我们的研究小组发现了hsp 90分子伴侣在血红素插入到三个关键位点中的特殊作用。 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 研究表明，强sGC-hsp 90相互作用可以是细胞中无血红素sGC的量度， 相互作用相对于sGC亚基异源二聚化是互斥的。总之，这些发现 在sGC功能失调的疾病状况的临床诊断中的潜在应用。我们发现 在炎症性哮喘中，sGC在一氧化氮（NO）升高的情况下变得功能失调， NO为基础的支气管扩张，但可以克服sGC激活剂，可以诱导支气管扩张，尽管 这种损失。哮喘中的这种sGC功能障碍与sGC功能障碍的强分子特征相关 其包含弱sGC-α1β1异二聚体、强sGCβ1-hsp 90相互作用和高S-亚硝基化 (SNO)对sGC-β1的影响我们目前和过去的研究表明，NO水平在生物学中至关重要， 这两种方法都能成功或失败sGC而哮喘中的高NO水平可以通过破坏sGC功能来诱导sGC功能障碍， sGC-α1β1异二聚体，低NO水平可触发sGC-β1中的血红素插入，增加和稳定sGC-α 1β1异二聚体， sGC异二聚体。此外，在人类哮喘ASMCs（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，我们提出（i）确定哮喘中sGC功能障碍的分子基础， 损害或保护sGC的细胞机制。这包括确定脱硝酶是否 如硫氧还蛋白-1（Trx-1）或NO清除剂Hb在根尖上皮中的表达可具有保护作用 气道平滑肌sGC (ii)在两种情况下建立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和肺组织样品。(iii)确定遗传、表观遗传和生化机制， 有缺陷的sGC (iv)探索在重度哮喘HASMC中恢复sGC功能的方法，包括治疗 NO暴露和过表达有益蛋白（Hsp 90，Trx-1，过氧化氢酶），其表达可能是 降低哮喘HASMCs。我们的项目将共同推进目前的知识如何伴侣， 氧化还原酶、NO和炎症调节健康和哮喘气道中的sGC，并提出了 恢复其功能。