GPx-3 and Peroxide Flux in the Endothelial Cell

描述（由申请人提供）：细胞氧化代谢导致活性氧（ROS）的产生。抗氧化机制已经发展到调节ROS的稳态通量并使其细胞毒性最小化。细胞的抗氧化潜力主要由低分子量还原剂和抗氧化酶控制。近年来，虽然血管细胞的ROS通量的细胞内决定因素已经得到了很好的表征，但ROS通量的细胞外决定因素及其调控还不太清楚。在该奖项的最后两个周期中，我们研究了一种调节细胞外过氧化物通量的关键抗氧化酶，谷胱甘肽过氧化物酶-3（GPx-3）。我们研究了这种硒蛋白的生物化学和分子细胞生物学，以了解其在调节内皮细胞一氧化氮的生物利用度和调节血管系统中血小板活化中的作用。虽然GPx-3可以有效地将过氧化氢和脂质过氧化物分别还原为水和脂质醇，但最近的动力学研究表明，过氧化氢是优选的底物。由于细胞外过氧化氢可以影响内皮细胞表面受体信号传导（低浓度）和细胞外氧化应激（高浓度），因此控制其产生，分子作用和血管系统中代谢命运的因素值得研究。该提议的中心假设是GPx-3是来自内皮细胞的细胞外过氧化氢通量的主要决定因素，并且是内皮细胞中过氧化氢的跨膜循环和作用的关键调节剂。为了验证这一假设，我们提出了三个具体目标。我们将首先评估GPx-3在消除过氧化氢中的作用，并将检查其在内皮细胞的细胞外微环境中的最佳活性所需的还原辅因子。其次，我们将确定过氧化氢在调节GPx-3的表达在转录，转录后和翻译水平的作用。第三，我们将研究GPx-3和其细胞内对应物GPx-1之间的相互作用对过氧化氢的跨细胞代谢和循环，以及其对内皮细胞中“由外向内”和“由内而外”信号传导的影响。这些研究应该提供洞察过氧化氢流量，细胞外抗氧化潜力，细胞信号和氧化应激在内皮细胞之间的复杂关系。

DESCRIPTION (provided by applicant): Cellular oxidative metabolism leads to the generation of reactive oxygen species (ROS). Antioxidant mechanisms have evolved to regulate the steady-state flux of ROS and minimize their cytotoxicity. The antioxidant potential of cells is largely governed by both low-molecular-weight reductants and by antioxidant enzymes. While the intracellular determinants of ROS flux by vascular cells have been well characterized in recent years, the extracellular determinants of ROS flux and their regulation are less well understood. For the last two cycles of this award, we have studied a key antioxidant enzyme that regulates extracellular peroxide flux, glutathione peroxidase-3 (GPx-3). We studied the biochemistry and molecular cell biology of this selenoprotein in order to understand its role in regulating the bioavailability of endothelial nitric oxide and in modulating platelet activation in the vasculature. While GPx-3 can effectively reduce both hydrogen peroxide and lipid peroxides to water and lipid alcohols, respectively, recent kinetic studies suggest that hydrogen peroxide is the preferred substrate. Because extracellular hydrogen peroxide can affect endothelial cell-surface receptor signaling (at low concentrations) and extracellular oxidant stress (at high concentrations), the factors that govern its production, molecular actions, and metabolic fates in the vasculature warrant study. The central hypothesis of this proposal is that GPx-3 is a principal determinant of extracellular hydrogen peroxide flux from the endothelial cell, and a key regulator of the transmembrane cycling and actions of hydrogen peroxide in the endothelial cell. To test this hypothesis, we propose three specific aims. We will first assess the role of GPx-3 in eliminating hydrogen peroxide and will examine the reductive cofactor(s) required for its optimal activity in the extracellular microenvironment of the endothelial cell. Second, we will determine the role of hydrogen peroxide in regulating the expression of GPx-3 at the transcriptional, posttranscriptional, and translational levels. Third, we will examine the interactions between GPx-3 and its intracellular counterpart GPx-1 on the transcellular metabolism and cycling of hydrogen peroxide, and its consequences for "outside-in" and "inside-out" signaling in endothelial cells. These studies should provide insight into the complex relationships among hydrogen peroxide flux, extracellular antioxidant potential, and cell signaling and oxidant stress in the endothelial cell.