MOLECULAR CHARACTERIZATION OF A LUNG SPHINGOMYELINASE

鞘磷脂酶是神经酰胺通路和肺活性氧化剂（如过氧化氢（H2）2）和过氧亚硝酸盐（ONOO-）)凋亡的调节因子，与肺上皮损伤和肺部疾病发病率增加密切相关。然而，将肺细胞暴露于氧化剂与肺部疾病发展联系起来的细胞和分子机制尚不清楚。我们之前的工作表明，氧化剂调节上游受体的功能，从而对气道上皮细胞的生长施加控制。最近，我们已经证明H2O2介导的氧化应激调节神经酰胺，细胞过程中的第二信使，诱导支气管上皮细胞凋亡。这些结果支持了我们的假设，即氧化应激、神经酰胺/鞘磷脂通路和诱导气道上皮细胞凋亡之间存在耦合。为了验证这一假设，我们将首先在细胞水平上表征氧化应激对神经酰胺途径的影响。我们将阐明相互作用的细胞位点，并确定哪种鞘磷脂酶（SMase）同工酶是由活性氧化剂调节以诱导细胞凋亡的。然后，我们将纯化并关闭在氧化应激和神经酰胺介导的细胞凋亡之间起耦合器作用的特异性SMase。这将使我们的研究进展从细胞到分子表征的机制(s)潜在的神经酰胺的产生和凋亡的调节。在细胞水平上表征氧化介导的神经酰胺生成，随后分离纯SMase蛋白和基因，是在细胞和分子水平上将该途径与肺损伤联系起来的重要里程碑。从长远来看，这一方向将为临床干预提供更精确的靶点，以控制肺上皮细胞的凋亡，从而防止肺上皮损伤这一肺部疾病的主要问题。

Molecular and Cellular Characterization of Sphingomyelinase, a Regulator of Ceramide Path and Apoptosis in the Lung Reactive oxidants, such as hydrogen peroxide (H2)2) and peroxynitrite (ONOO-), are strongly associated with lung epithelium injury and with the increased incidence of lung disease. Yet, the cellular and molecular mechanisms that link exposure of lung cells to oxidants with the development of lung disease are poorly understood. Our previous work has shown that oxidants modulate the function of upstream receptors and therefore exert growth control on airway epithelial cells. Recently, we have shown that H2O2- mediated oxidative stress modulates ceramide, a second messenger in cellular processes, to induce apoptosis in the bronchial epithelium. These results support our hypothesis that there is coupling between oxidative stress, the ceramide/sphingomyelin pathway, and induction of apoptosis in airway epithelial cells. To test this hypothesis, we will first characterize the effects of oxidative stress on the ceramide pathway at the cellular level. We will elucidate the cellular sites of interaction and determine which sphingomyelinase (SMase) isozyme is regulated by reactive oxidants to induce apoptosis. Then, we will purify and cloe the specific SMase which acts as the coupler between oxidative stress and ceramide-mediated apoptosis. This will allow our studies to progress from cellular to molecular characterization of the mechanism(s) underlying the regulation of ceramide generation and apoptosis. Characterization of oxidant-mediated ceramide generation at the cellular level, followed by isolation of the pure SMase protein and gene are important milestones that would link this pathway to lung injury at the cellular and molecular levels. In the long run, this direction will lead to more precise targets for clinical intervention to control apoptosis in lung epithelial cells, thus preventing epithelial injury, a major problem in lung disease.