MOLECULAR CHARACTERIZATION OF A LUNG SPHINGOMYELINASE

肺神经酰胺通路和细胞凋亡调节因子鞘磷脂酶的分子和细胞特性活性氧化剂，如过氧化氢（H2）2）和过氧亚硝酸盐（ONOO-），与肺上皮损伤和肺部疾病发病率的增加密切相关。 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，因此对气道上皮细胞的生长控制。 最近，我们已经表明，过氧化氢介导的氧化应激调节神经酰胺，在细胞过程中的第二信使，诱导细胞凋亡的支气管上皮。 这些结果支持了我们的假设，氧化应激，神经酰胺/鞘磷脂途径，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描述氧化应激对神经酰胺通路的影响。 我们将阐明相互作用的细胞位点，并确定哪些鞘磷脂酶（SMase）同工酶受反应性氧化剂的调节，以诱导细胞凋亡。 在此基础上，我们将对神经酰胺介导的细胞凋亡与氧化应激之间的偶联物SMase进行纯化和克隆。 这将使我们的研究从神经酰胺生成和细胞凋亡调节机制的细胞到分子表征。在细胞水平上对氧化剂介导的神经酰胺生成进行表征，然后分离纯SMase蛋白和基因，是将该途径与细胞和分子水平上的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.