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)受到多种转录、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，并且这种关联增加了eNOS的活性。我们提出了一个新的假设，即eNOS-肌动蛋白结合调节eNOS的电子传递，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eNOS的调节。目的#1：确定肌动蛋白是否与eNOS蛋白中可能的肌动蛋白结合序列(ABSS)相互作用，从而增加eNOS的电子传递和催化活性。我们将使用酵母双杂交系统和含有eNOS三个假定ABS序列的多肽来定位eNOS蛋白中的肌动蛋白结合部位。我们将评估eNOS的ABS多肽对eNOS活性、eNOS-肌动蛋白相互作用、酶动力学的影响，以及对纯化的eNOS和肌动蛋白混合物中细胞色素c和铁氰化钾还原酶活性的影响，以阐明通过肌动蛋白结合提高eNOS活性的机制。目的#2：确定不同氧分压下肺内皮细胞eNOS活性的改变是否源于eNOS-肌动蛋白结合的改变。我们将利用siRNA和腺病毒基因转移技术控制内皮细胞中肌动蛋白的可获得性，并通过显微注射技术将eNOS的ABS多肽导入内皮细胞以阻断eNOS-肌动蛋白的相互作用。然后，我们将观察低氧/高氧诱导的eNOS活性、肌动蛋白细胞骨架、eNOS-肌动蛋白结合、NO释放和内皮依赖性血管松弛的变化。这些目标的成功实现将促进我们对NO生物学的理解，进而对以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。确定短期高氧治疗的效果将有助于我们加深对氧疗机制的理解，并将有助于更好地管理接受补充氧疗的患者。 公共卫生相关性：这项建议是为了研究一种新的假说，即eNOS-肌动蛋白结合调节eNOS的电子传递，从而导致催化活性增加，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eNOS的调节。证实eNOS-肌动蛋白相互作用在低氧/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，进而对以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、慢性阻塞性肺疾病和肺心病具有重要意义。确定短期高氧治疗的效果将有助于我们加深对氧疗机制的理解，并将有助于更好地管理接受补充氧疗的患者。

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.