eNOS-actin Interaction and Oxygen in Lung Endothelium

描述（申请人提供）：内皮型一氧化氮合酶（eNOS）受多种转录、转录后和翻译后机制的严格调控。我们已经发现eNOS与肌动蛋白相关，并且这种关联增加eNOS活性。我们提出了一个新的假说，eNOS-肌动蛋白协会调节eNOS的电子转移，导致催化活性增加，eNOS-肌动蛋白相互作用的改变介导了不同氧分压下肺内皮细胞中eNOS的调节。目标一：确定肌动蛋白是否与eNOS蛋白中假定的肌动蛋白结合序列（ABS）相互作用，导致eNOS的电子转移和催化活性增加。我们将使用酵母双杂交系统和具有eNOS的三个推定ABS序列的肽来精确定位eNOS蛋白中的肌动蛋白结合位点。我们将评估eNOS的ABS肽对eNOS活性、eNOS-肌动蛋白相互作用、酶动力学以及纯化的eNOS和肌动蛋白的混合物中的细胞色素c和铁氰化物还原酶活性的影响，以阐明通过肌动蛋白缔合增加eNOS活性的机制。目标二：确定暴露于不同氧分压的肺内皮细胞中eNOS活性的改变是否是由于eNOS-肌动蛋白结合的变化。我们将利用siRNA和腺病毒基因转移技术来调控内皮细胞中肌动蛋白的可利用性，并利用显微注射技术将eNOS的ABS肽段导入内皮细胞，阻断eNOS与肌动蛋白的相互作用。然后，我们将观察缺氧/高氧诱导的eNOS活性，肌动蛋白细胞骨架，eNOS-肌动蛋白协会，NO释放和内皮依赖性血管舒张的变化。这些目标的成功完成将推进我们对NO生物学的理解，反过来，对以eNOS活性受损和血管NO产生为特征的肺部疾病，如原发性和继发性肺动脉高压、COPD和肺心病具有重大意义。确定短期高氧的影响将有助于我们进一步了解氧疗的机制，并将导致更好的管理策略，为患者接受补充氧疗。 公共卫生相关性：本研究提出了一个新的假说，即eNOS-肌动蛋白结合调节eNOS的电子传递，导致催化活性增加，并且eNOS-肌动蛋白相互作用的改变介导了暴露于不同氧分压下的肺内皮细胞中eNOS的调节。证明eNOS-肌动蛋白相互作用在缺氧/高氧诱导的eNOS功能改变中的作用将促进我们对NO生物学的理解，并且反过来，对以eNOS活性受损和血管NO产生为特征的肺部疾病（如原发性和继发性肺动脉高压、COPD和肺心病）具有重要意义。确定短期高氧的影响将有助于我们进一步了解氧疗的机制，并将导致更好的管理策略，为患者接受补充氧疗。

DESCRIPTION (provided by applicant): Endothelial nitric oxide synthase (eNOS) is tightly regulated by a variety of transcriptional, posttranscriptional, and posttranslational mechanisms. We have found that eNOS is associated with the actin protein and that this association increases eNOS activity. We propose a novel hypothesis that eNOS-actin association regulates electron transfer of eNOS resulting in increased catalytic activity and that alterations in eNOS-actin interaction mediate the regulation of eNOS in lung endothelium exposed to different oxygen tensions. Aim #1: Determine whether actin interacts with the putative actin binding sequences (ABSs) in the eNOS protein resulting in increased electron transfer and catalytic activity of eNOS. We will use a yeast two-hybrid system and the peptides with sequences of the three putative ABSs of eNOS to pinpoint the actin-binding site in the eNOS protein. We will evaluate the effects of ABS peptides of eNOS on eNOS activity, eNOS-actin interaction, the enzyme kinetics, and on cytochrome c and ferricyanide reductase activities in the mixture of purified eNOS and actin to clarify the mechanism for increased eNOS activity by actin association. Aim #2: Determine whether alterations of eNOS activity in lung endothelium exposed to different oxygen tensions are due to changes in eNOS-actin association. We will manipulate the availability of actin in endothelium using siRNA and adenovirus gene transfer technology and introduce the ABS peptides of eNOS into endothelial cells to block eNOS-actin interaction using microinjection technique. Then we will observe hypoxia/hyperoxia-induced alterations in eNOS activity, the actin cytoskeleton, eNOS-actin association, NO release, and endothelium-dependent vasorelaxation. The successful completion of these aims will advance our understanding of the biology of NO, and in turn, have major implications for pulmonary diseases characterized by impaired eNOS activity and vascular NO production, such as primary and secondary pulmonary hypertension, COPD, and cor pulmonale. Determination of the effect of short-term hyperoxia will help to advance our understanding of the mechanism of oxygen therapy and will lead to better management strategy for patients receiving supplemental oxygen therapy. PUBLIC HEALTH RELEVANCE: This proposal is to study a novel hypothesis that eNOS-actin association regulates electron transfer of eNOS resulting in increased catalytic activity and that alterations in eNOS-actin interaction mediate the regulation of eNOS in lung endothelium exposed to different oxygen tensions. Proof of the role of eNOS-actin interaction in hypoxia/hyperoxia-induced alteration of eNOS function would advance our understanding of the biology of NO, and in turn, have major implications for pulmonary diseases characterized by impaired eNOS activity and vascular NO production, such as primary and secondary pulmonary hypertension, COPD, and cor pulmonale. Determination of the effect of short-term hyperoxia will help to advance our understanding of the mechanism of oxygen therapy and will lead to better management strategy for patients receiving supplemental oxygen therapy.