Molecular Characteriszation of a Novel Lung Sphingomyelinase

描述(申请人提供)：不受控制的神经酰胺信号导致不受控制的细胞凋亡，这是哮喘、支气管扩张和慢性阻塞性肺疾病等肺部疾病中肺损伤发病机制的一个标志。我们的研究表明，活性氧物种(ROS)是神经酰胺生成和细胞凋亡的关键参与者。然而，目前关于氧化剂暴露与神经酰胺和细胞凋亡，并最终与肺损伤的发展有关的细胞和分子机制还很少。我们已经证明，暴露在ROS下的人呼吸道上皮细胞会产生过量的神经酰胺，神经酰胺是这些细胞中细胞死亡的有效诱导剂。因此，我们在之前的资助期间提出，神经酰胺的增加是肺部疾病所观察到的细胞损伤的主要原因。我们对ROS和神经酰胺产生之间的联系的研究产生了一种新的中性鞘磷脂酶nSMase2，它是从猴肺组织和人呼吸道上皮细胞中分离出来的。我们的初步研究表明，nSMase2是神经鞘磷脂酶的主要靶标(神经鞘磷脂酶的一个大家族)，被氧化剂激活，并诱导神经酰胺的产生上调，导致细胞死亡和肺损伤。在将nSMase2与氧化应激和神经酰胺诱导的肺损伤联系在一起后，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/肺泡破坏中细胞死亡的关键开关。为了证明这一假设，我们目前的建议试图探索和定义nSMase2激活的细胞、分子和生化机制，并通过香烟烟雾暴露在小鼠或大鼠的肺损伤模型来证明其在体内的作用。在具体目标1中，我们将研究nSMase2在原代人和猴呼吸道上皮细胞以及暴露于CS诱导的氧化应激下的小鼠[或大鼠]产生神经酰胺和诱导细胞死亡中的作用。在特定的目标2中，我们将确定哪些蛋白质-蛋白质相互作用参与了nSMase2的功能、细胞定位和激活及其在氧化应激下的调节。最后，在特定的目标3中，我们将定义nSMase2的结构和功能，描述氧化应激如何导致其高活性半胱氨酸残基、特定的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的蛋白质相互作用有关。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，特别是它在细胞死亡和上皮/肺泡破坏中的作用。这些研究的成功将开辟新的治疗途径，针对肺损伤和肺部疾病中nSMase2驱动的过程。 公共卫生相关性：我们目前的研究集中在以小鼠为模型生物，描述参与新的nSMAse2激活的细胞、分子和生化机制及其在体内肺损伤中的作用。这些研究的成功将开辟新的治疗途径，针对肺损伤和肺部疾病中nSMase2驱动的过程。

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.