MOLECULAR CHARACTERIZATION OF A LUNG SPHINGOMYELINASE

神经鞘磷脂酶是一种调节神经酰胺途径和肺内细胞凋亡的活性氧化剂，如过氧化氢(H2)2和过氧亚硝酸根(ONOO-)，其分子和细胞特性与肺上皮损伤和肺部疾病的增加密切相关。然而，将肺细胞暴露于氧化剂与肺部疾病的发展联系起来的细胞和分子机制还知之甚少。我们以前的工作表明，氧化剂调节上游受体的功能，从而对呼吸道上皮细胞施加生长控制。最近，我们发现过氧化氢介导的氧化应激调节细胞过程中的第二信使神经酰胺，诱导支气管上皮细胞凋亡。这些结果支持我们的假设，即氧化应激、神经酰胺/鞘磷脂途径和诱导呼吸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。我们将阐明细胞内相互作用的部位，并确定哪些鞘磷脂酶(SMase)同工酶是由反应性氧化剂调节的，以诱导细胞凋亡。然后，我们将纯化并关闭特定的sMase，它在氧化应激和神经酰胺介导的细胞凋亡之间起着偶联作用。这将使我们的研究从细胞到分子表征神经酰胺生成和细胞凋亡的调控机制(S)。在细胞水平上鉴定氧化剂介导的神经酰胺生成，然后分离纯化的sMase蛋白和基因是在细胞和分子水平上将这一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准确的靶点，以控制肺上皮细胞的凋亡，从而防止上皮损伤，这是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.