Redox Regulation of Src Kinase in Allergic Airway Inflammation

项目摘要 原癌基因酪氨酸激酶Src是一种广泛表达的非受体酪氨酸激酶， 参与许多关键的细胞功能，如迁移、增殖和存活。虽然异常的Src 虽然信号传导与癌症生物学有关，但它也有助于非恶性疾病，如哮喘。 Src的典型调节涉及酪氨酸磷酸化和与调节蛋白的变构相互作用， 但最近的证据表明，保守的半胱氨酸残基的氧化也在Src活化中起作用， 与下游目标的相互作用。在气道上皮中，对损伤或空气传播的先天反应 过敏原暴露被发现涉及表皮生长因子受体（EGFR）的反式激活， 与Src半胱氨酸氧化有关。我们的初步数据表明，Src是关键的激活型 2对这些触发物的先天免疫应答，并且Src的氧化活化由NADPH介导。 氧化酶双氧化酶1（DUOX 1）。然而，Src含有9个半胱氨酸残基， 在调节Src活化中，靶结合和亚细胞定位是未知的。而且，不得而知 这些氧化还原过程如何影响过敏原诱导的2型反应或过敏性哮喘的发展。我们 假设变应原介导DUOX 1依赖性Src半胱氨酸氧化对于增强IL-33是关键的 释放和激活2型免疫应答。为了解决这一假设，我们将使用体外和体内 利用Src缺陷小鼠或基于RNAi的方法沉默Src的过敏原暴露模型。我们将确定 Src的氧化还原调节对2型免疫应答和过敏性哮喘发生的影响 评估IL-33的分泌和表达，下游2型应答的激活，以及过敏反应的特征。 屋尘螨诱导过敏性哮喘模型中的炎症。半胱氨酸的功能后果 Src活化或下游信号传导活化的氧化将使用各种Cys-至-Ala Src的变体转染到肺上皮细胞中。总的来说，我们的研究将揭示特定的 变应性哮喘背景下Src活化的氧化还原改变。

Project Summary The proto-oncogene tyrosine kinase Src is a widely expressed non-receptor tyrosine kinase that is involved in many critical cellular functions such as migration, proliferation, and survival. Although aberrant Src signaling has been implicated in cancer biology, it also contributes to non-malignant diseases such as asthma. Canonical regulation of Src involves tyrosine phosphorylation and allosteric interactions with regulatory proteins, but recent evidence has shown that oxidation of conserved cysteine residues also plays a role in Src activation and interactions with downstream targets. In the airway epithelium, innate responses to injury or airborne allergen exposure were found to involve transactivation of the epidermal growth factor receptor (EGFR) in association with Src cysteine oxidation. Our preliminary data demonstrate that Src is critical for activation of type 2 innate-immune responses to these triggers, and that oxidative activation of Src is mediated by the NADPH oxidase dual oxidase 1 (DUOX1). However, Src contains 9 cysteine residues and the specific cysteines involved in regulating Src activation, target binding and subcellular localization are unknown. Moreover, it is not known how these redox processes impact on allergen-induced type 2 responses or development of allergic asthma. We hypothesize that allergen-mediated DUOX1-dependent Src cysteine oxidation is critical for enhanced IL-33 release and activation of type 2 immune responses. To address this hypothesis, we will use in vitro and in vivo models of allergen exposure utilizing Src-deficient mice or RNAi-based methods to silence Src. We will determine the impact of redox regulation on Src on type 2 immune responses and the development of allergic asthma by assessing secretion and expression of IL-33, activation of downstream type 2 responses, and features of allergic inflammation in a house dust mite-induced model of allergic asthma. The functional consequence of cysteine oxidation for Src activation or activation of downstream signaling will be addressed using various Cys-to-Ala variants of Src transfected into lung epithelial cells. Collectively, our studies will reveal the importance of specific redox alterations in Src activation in the context of allergic asthma.