S-nitrosylation signaling in asthma

项目总结/摘要 项目1 哮喘折磨着超过7%的美国人，是气道慢性炎症导致气道炎症的结果。 重塑和高反应性，阻碍空气流动。严重哮喘是哮喘仍然存在问题 尽管常规哮喘治疗进行了最大限度的干预，但它仍占大多数死亡率， 哮喘的成本。气道β2-肾上腺素能受体（β 2-adrenergic receptor，β2AR）信号转导功能障碍与严重哮喘的关系 发病机制，但确切的信号机制尚不清楚。当β 2 AR被激活时， 吸入性“β-激动剂”药物是急性哮喘治疗的主要手段，β2AR的过度激活是有害的， 是致命的。气道β 2AR通过异源三聚体G蛋白和G蛋白偶联受体信号传导 激酶（GRK）/β-抑制蛋白途径，也介导受体磷酸化，脱敏， 内化通过长期合作，Stamler和Gaston团队发现， 通过硫醇的S-亚硝基化形成S-亚硝基硫醇（SNO），由一氧化氮（NO）调节，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 在低分子量硫醇上形成，包括谷胱甘肽以形成SNO-谷胱甘肽（GSNO）。我们证明 吸入GSNO可以提高肺蛋白-SNO，对哮喘有保护作用， 谷胱甘肽还原酶（GSNOR）使GSNO失活，并证明缺乏GSNOR的小鼠 防止哮喘的发生。由于β2AR可以激活气道中的NO合酶产生NO， 需要发现其如何促进内源性SNO介导的支气管保护作用。我们最近的工作 已经表明β2AR在激活后被S-亚硝基化，用点突变阻止SNO-β2AR增加 β2AR信号传导。重要的是，携带β2AR并敲入该突变的小鼠可以避免发展成 哮喘我们先前已经证明β2AR调节剂GRK 2和β-arrestin 2被S-亚硝基化以抑制它们的表达。 GRK 2-C340 S和β-arrestin 2-C253 S基因敲入的小鼠表现出更高的β 2 AR脱敏活性， 心脏模型中β 2 AR活性和加重的损伤。我们已经开始测试吸入GSNO的功效， 影响患者的支气管舒张和改善肺功能，这些数据和临床样本 使我们能够从实验室到床旁检查S-亚硝基化在调节β2AR通路中的作用。的 项目1的中心假设是β2AR信号系统是SNO-GSNO-1的关键靶点和介导者。 GSNOR在严重哮喘气道中的保护作用。我们的研究将确定S-亚硝基化的作用， 哮喘小鼠模型中特异性β2AR信号通路成分，描述吸入GSNO的作用 在哮喘小鼠模型和人肺中GSNO二亚硝基化酶对β 2 AR信号成分的影响 原代细胞，并证明吸入GSNO改善气道流量和β2-激动剂反应性， 严重哮喘这项工作通过提供一种机械联系来补充项目2和3中的临床目标 NO/GSNO/GSNOR作用与β 2 AR信号调节之间的关系

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.