Estrogen and Endothelial Protein Nitrosylation

描述（由申请人提供）：通过向半胱氨酸添加一氧化氮（NO）衍生的亚硝酰基形成S-亚硝基硫醇，调节多种蛋白质的功能。这种翻译后蛋白质修饰被命名为S-亚硝基化（S-NO），其生物学意义被认为类似于磷酸化。它代表了一种新兴的生理信号机制，即NO直接影响蛋白质及其功能。在这个修订的R21申请中，我们的目的是鉴定新的内皮细胞和线粒体SNO蛋白，并通过使用原代子宫动脉内皮细胞和人脐静脉内皮细胞作为模型，确定SNO-cofilin-1的功能后遗症和与ER和内源性NO通过eNOS相关的机制。目标1：采用CyDye Switch、二维荧光差异凝胶电泳（2D-DIGE）和基质辅助激光解吸/电离飞行时间（MALDI/TOF）质谱分析雌激素反应性内皮细胞亚硝酰蛋白质组。目标二：通过CyDye Switch、2D-DIGE和MALDI-TOF技术分析E22诱导的内皮细胞线粒体亚硝酰基蛋白质组，并确定E22诱导的内皮细胞亚硝酰基蛋白质的亚细胞定位。目标3：确定雌激素诱导的蛋白质如cofilin-1的S-NO是否由特异性ER和内源性NO通过eNOS介导的机制介导。目的4：研究雌激素对内皮细胞肌动蛋白重组和迁移的影响。本研究建立在广泛的体外和体内研究基础上，这些研究表明雌激素对内皮eNOS表达和NO产生具有刺激作用;然而，所提出的研究是第一个采取下一步来确定通过雌激素刺激增加NO产生直接影响蛋白质及其功能的下游细胞和生理过程，因此在雌激素中具有关键的生物学意义，NO和内皮生物学。这些研究在围产期医学中是重要的，因为内皮/NO依赖性血管舒张是负责雌激素诱导的和妊娠相关的子宫血流量升高的关键机制，其与胎儿发育/存活和围产期/新生儿结局直接相关。这项研究也将促进我们对雌激素在心血管系统中的保护作用的理解。 公共卫生相关性：该R21旨在分析内皮细胞和线粒体亚硝基蛋白质组，希望确定新的雌激素反应性亚硝基化蛋白质靶点，并研究cofilin-1亚硝基化在内皮细胞肌动蛋白组织/细胞迁移中的作用。该研究采取了下一步的第一步，以确定通过雌激素刺激增加一氧化氮产生直接影响蛋白质及其功能的下游事件。所获得的数据将提供知识飞跃，了解雌激素诱导的和妊娠相关的子宫血管舒张对胎儿发育/存活和围产期/新生儿结局至关重要，也与雌激素保护心血管系统有关。

DESCRIPTION (provided by applicant): Formation of S-nitrothiols via addition of nitric oxide (NO)-derived nitrosyl groups to cysteines regulates the function of a plethora of proteins. This post-translational protein modification has been named as S- nitrosylation (S-NO) whose biological significance has been proposed to be analogous to phosphorylation. It represents an emerging physiological signaling mechanism that NO directly affects proteins and their functions. In this revised R21 application we are aimed at identifying novel endothelial cellular and mitochondrial SNO-proteins and at determining the functional sequelae of SNO-cofilin-1 and mechanisms linked to ER and endogenous NO via eNOS by using primary uterine artery endothelial cells and human umbilical vein endothelial cells as the models. Aim 1: To analyze estrogen responsive endothelial cellular nitrosyl-proteome by CyDye Switch, 2D two-dimensional fluorescence difference gel electrophoresis (2D- DIGE) and matrix-assisted laser desorption/ionization-time of flight (MALDI/TOF) mass spectrometry. Aim 2: To determine the subcellular localization of SNO-proteins in response to E22 and to analyze endothelial mitochondrial nitrosyl-proteome by CyDye Switch, 2D-DIGE and MALDI-TOF. Aim 3: To determine if estrogen-induced S-NO of proteins such as cofilin-1 is mediated by specific ER and endogenous NO via eNOS mediated mechanism(s). Aim 4: To determine if SNO-cofilin-1 regulates endothelial cell actin reorganization and migration by estrogen. This research is built on extensive in vitro and in vivo studies showing a stimulatory effect of estrogens on endothelial eNOS expression and NO production; however, the proposed studies are the first to take the next step for determining the downstream cellular and physiological processes that increased NO production by estrogen stimulation directly affects proteins and their functions and thus are of critical biological significance in estrogen, NO and endothelial biology. These studies are important in perinatal medicine as endothelium/NO dependent vasodilatation is a key mechanism responsible for estrogen-induced and pregnancy-associated rises in uterine blood flow that directly correlates to fetal development/survival and perinatal/neonatal outcomes. This research also will advance our understanding of the protective effects of estrogens in the cardiovascular system. PUBLIC HEALTH RELEVANCE: This R21 aims to analyze endothelial cellular and mitochondrial nitrosyl-proteomes in hopes of identifying novel estrogen responsive nitrosylated protein targets and to investigate the role of cofilin-1 nitrosylation in endothelial cell actin organization/cell migration. The research takes the first next step for determining the downstream events that increased nitric oxide production by estrogen stimulation directly affects proteins and their functions. Data gained will provide knowledge leaps in understanding estrogen-induced and pregnancy associated uterine vasodilation critical for fetal development/survival and perinatal/neonatal outcomes and also relevant to estrogen protection of the cardiovascular system.