Molecular Characteriszation of a Novel Lung Sphingomyelinase

描述（由申请人提供）：神经酰胺信号不调节导致细胞凋亡失控，这是肺部疾病（如哮喘、支气管扩张和慢性阻塞性肺疾病）肺损伤发病机制的一个标志。我们的研究表明活性氧（ROS）是神经酰胺生成和细胞凋亡的关键参与者。然而，目前关于氧化暴露与神经酰胺和细胞凋亡之间的细胞和分子机制，以及最终与肺损伤的发展之间的联系，我们知之甚少。我们已经证明，暴露于ROS的人气道上皮细胞产生过量的神经酰胺，神经酰胺在这些细胞中作为细胞死亡的有效诱导剂。因此，我们在之前的资助期内提出，神经酰胺增加是肺部疾病中观察到的细胞损伤的主要原因。我们对活性氧和神经酰胺产生之间联系的研究发现了一种新的中性鞘磷脂酶nSMase2，该酶从猴肺组织和人气道上皮细胞中分离出来。我们的初步研究表明，nSMase2是鞘磷脂酶的主要靶点（鞘磷脂酶（SMases）大家族中的一员），它被氧化暴露激活，诱导神经酰胺产生上调，导致细胞死亡和肺损伤上调。在将nSMase2与氧化应激和神经酰胺诱导的肺损伤联系起来之后，我们现在假设nSMase2实际上是启动和调节神经酰胺诱导的肺上皮/肺泡破坏中的细胞死亡的关键开关。为了证明这一假设，我们目前的建议旨在探索和定义nSMase2激活的细胞、分子和生化机制，并通过小鼠或大鼠的香烟烟雾（CS）暴露肺损伤模型来证明其在体内的作用。在Specific Aim 1中，我们将研究nSMase2在神经酰胺生成和诱导细胞死亡中的作用，使用暴露于cs诱导的氧化应激的人类和猴子气道上皮细胞以及小鼠[或大鼠]。在Specific Aim 2中，我们将确定哪些蛋白-蛋白相互作用参与了nSMase2的功能、细胞定位和激活及其在氧化应激下的调节。最后，在Specific Aim 3中，我们将定义nSMase2的结构-功能，描述氧化应激如何导致其高活性Cys残基、特定磷酸化位点和泛素化的变化，以及这些变化如何与nSMase2与神经酰胺信号通路中蛋白质的相互作用相关联。通过这些综合研究，我们将阐明新的nSMase2调控途径的生物学意义和分子机制，特别是其在细胞死亡和上皮/肺泡破坏中的作用。这些研究的成功将为针对nsmase2驱动的过程治疗肺损伤和肺部疾病开辟新的治疗途径。

DESCRIPTION (provided by applicant): Unregulated ceramide signaling causes uncontrolled cellular apoptosis, a hallmark in the pathogenesis of lung injury in pulmonary diseases such as asthma, bronchiectasis, and chronic obstructive pulmonary disorders. Our studies have demonstrated that reactive oxygen species (ROS) are key participants in ceramide generation and apoptosis. Yet, currently there is little insight regarding the cellular and molecular mechanisms linking oxidant exposure with ceramide and apoptosis, and ultimately with development of lung injury. We have shown that human airway epithelial cells exposed to ROS generate excessive ceramide, which functions as a potent inducer of cell death in these cells. We therefore proposed under the previous funding period that increased ceramide is a primary cause of the cellular injury observed with lung diseases. Our search for the link between ROS and ceramide production yielded a novel neutral sphingomyelinase, nSMase2, which was isolated from monkey lung tissue and human airway epithelial cells. Our preliminary studies suggested that nSMase2 is the main target sphingomyelinase (out of a large family of sphingomyelinases (SMases)) that is activated by oxidant exposure, and induces upregulated ceramide production, leading to upregulated cell death and lung injury. Having linked nSMase2 to oxidative stress and ceramide-induced lung injury, we now hypothesize that nSMase2 is in fact a crucial switch that initiates and regulates the ceramide-induced cellular death in lung epithelial/alveolar destruction. To prove this hypothesis, our current proposal seeks to explore and define the cellular, molecular, and biochemical mechanisms of nSMase2 activation and also to demonstrate its role in vivo using a lung injury model of cigarette smoke (CS) exposure in mice or rats. In Specific Aim 1, we will investigate the role of nSMase2 in ceramide generation and induction of cell death using primary human and monkey airway epithelial cells as well as mice [or rats] exposed to CS-induced oxidative stress. In Specific Aim 2, we will determine which protein-protein interactions are involved in the function, cellular localization and activation of nSMase2 and its modulation under oxidative stress. Finally, in Specific Aim 3, we will define the structure-function of nSMase2, characterizing how oxidative stress leads to changes in its hyper-reactive Cys residues, specific phosphorylation sites, and ubiquitinylation, and how these changes may be linked to nSMase2 interactions with proteins in the ceramide signaling pathway. Through these comprehensive studies, we will elucidate the biological significance and molecular mechanisms of the novel nSMase2 regulatory pathway, specifically its role in cell death and epithelial/alveolar destruction. Success of these studies will open new therapeutic avenues that target nSMase2-driven processes in lung injury and pulmonary diseases. PUBLIC HEALTH RELEVANCE: Our present studies focus on the characterization of the cellular, molecular and biochemical mechanisms involved in the activation of the novel nSMAse2 and its roles in lung injury in vivo using mice as a model organism. Success of these studies will open new therapeutic avenues that target nSMase2-driven processes in lung injury and pulmonary diseases.