S-nitrosylation signaling in asthma

项目摘要/摘要 项目1 超过7%的美国人患有哮喘，它是导致呼吸道慢性炎症的结果 阻碍气流的重塑和高反应性。严重哮喘是仍有问题的哮喘 尽管用传统的哮喘疗法进行了最大限度的干预，但它占了死亡的大部分 以及哮喘的费用。气道β2肾上腺素能受体(β2AR)信号转导功能障碍与重症哮喘的关系 发病机制尚不清楚，但相关的确切信号机制尚不清楚。在使用以下命令激活β2AR时 吸入β激动剂药物是急性哮喘治疗的主要手段，β2AR过度激活是有害的，可以 是致命的。呼吸道β2ARs通过异源三聚体G蛋白和G蛋白偶联受体传递信号 激酶(Grk)/β-arrestin通路也介导受体的磷酸化、脱敏和 内部化。通过长期的合作，Stamler和Gaston团队发现Airways 受一氧化氮(NO)调节，通过硫醇的S-亚硝化生成S-亚硝硫醇(SNO)，包括 蛋白质中的半胱氨酸残基，一种改变蛋白质功能的翻译后修饰。此外，SNO 形成低分子量硫醇，包括谷胱甘肽，形成亚硝基-谷胱甘肽(GSNO)。我们演示了 吸入GSNO可升高肺蛋白-SNO，对哮喘具有保护作用，鉴定了SNO-1酶 谷胱甘肽还原酶(GSNOR)使GSNO失活，并证明缺乏GSNOR的小鼠 防止发展成哮喘。由于β2AR可以激活呼吸道中的NO合成酶来产生NO， 有必要发现这是如何促进内源性SNO介导的支气管保护的。我们最近的工作 已经证明β-β2AR在激活后是S亚硝化的，并通过点突变阻止SNO-SNO-2AR的增加 β2AR信令。重要的是，携带β2AR基因突变敲入的小鼠可以免受疾病侵袭 哮喘。我们之前已经证明，β2AR调节剂grk2和β-arrestin2是S硝化的，以抑制他们的 β2AR的脱敏活性以及携带GRK2-C340S和β-arrestin2-C253S敲入的小鼠表现出更高的活性 β-2AR活性升高，心肌损伤加重。我们已经开始测试吸入GSNO的疗效 影响患者的支气管松弛和改善肺功能，这些数据和临床样本是独一无二的 让我们来研究S-亚硝化在调节β-2AR通路中的作用。这个 项目1的中心假设是β2AR信号系统是SNO-GSNO-2的关键靶点和介体。 GSNOR对重症哮喘患者呼吸道的保护作用。我们的研究将确定S的作用-亚硝化 哮喘小鼠模型中特异的β-2AR信号通路成分--吸入GSNO的作用 哮喘小鼠和人肺组织中GSNO二硝基转移酶对β-2AR信号成分的影响 并证明吸入GSNO可改善气道流量和β-2激动剂的反应性。 严重的哮喘。这项工作通过提供一种机械链接来补充项目2和3中的临床目标 重症哮喘患者NO/GSNO/GSNOR作用与β-2AR信号调节的关系

PROJECT SUMMARY/ABSTRACT Project 1 Asthma afflicts over 7% of Americans and is the result of chronic inflammation of the airways leading to airway remodeling and hyperresponsiveness that impedes air flow. Severe asthma is asthma that remains problematic despite maximal intervention with conventional asthma therapies, and it accounts for the majority of the mortality and cost of asthma. Dysfunction of airway β2-adrenergic receptor (β2AR) signaling contributes to severe asthma pathogenesis, but the precise signaling mechanisms responsible are unclear. While activation of β2AR using inhaled “β-agonist” drugs is a mainstay of acute asthma treatment, overactivation of β2AR is detrimental and can be fatal. Airway β2ARs signal both through heterotrimeric G proteins and through G protein-coupled receptor kinase (GRK)/β-arrestin pathways that also mediate receptor phosphorylation, desensitization, and internalization. Through a long-standing collaboration, the Stamler and Gaston groups have found that airways are regulated by nitric oxide (NO) through S-nitrosylation of thiols to form S-nitrosothiol (SNO), including on cysteine residues in proteins, a post-translational modification that alters protein functions. In addition, SNO forms on low molecular weight thiols, including glutathione to form SNO-glutathione (GSNO). We demonstrated that inhaled GSNO elevates lung protein-SNO and is protective in asthma, identified the enzyme SNO- glutathione reductase (GSNOR) that inactivates GSNO, and demonstrated that mice lacking GSNOR are protected from developing asthma. Since the β2AR can activate NO synthase in the airways to generate NO, there is a need to discover how this promotes endogenous SNO-mediated bronchoprotection. Our recent work has shown that β2AR is S-nitrosylated after activation, and preventing SNO-β2AR with a point mutation augments β2AR signaling. Importantly, mice bearing β2AR with a knock-in of this mutation are protected from developing asthma. We have previously shown that β2AR regulators GRK2 and β-arrestin2 are S-nitrosylated to inhibit their activity to desensitize the β2AR, and mice bearing GRK2-C340S and β-arrestin2-C253S knock-in exhibit heighted β2AR activity and worsened injury in cardiac models. We have begun to test the efficacy of inhaled GSNO to affect bronchorelaxation and improved lung function in patients, and these data and clinical samples uniquely position us to examine the role of S-nitrosylation in regulating the β2AR pathway from bench to bedside. The Central Hypothesis of Project 1 is that the β2AR signaling system is a key target and mediator of SNO-GSNO- GSNOR protective effects in the airways in severe asthma. Our studies will define the role of S-nitrosylation of specific β2AR signaling pathway components in a murine model of asthma, delineate the roles of inhaled GSNO and of GSNO dinitrosylases on β2AR signaling components in murine models of asthma and in human lung primary cells, and demonstrate that inhaled GSNO improves both airway flow and β2-agonist responsiveness in severe asthma. This work complements the clinical aims in Projects 2 and 3 by providing a mechanistic link between NO/GSNO/GSNOR actions and the regulation of β2AR signaling in severe asthma.