Estrogen and Endothelial Protein Nitrosylation

雌激素和内皮蛋白亚硝基化

• 批准号: 7991392
• 负责人: DONGBAO CHEN
• 金额: $ 22.95万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7991392
• 关键词: Actin-Binding Protein; Actins; Affect; Anabolism; Apoptosis; Arteries; Attention; Biochemical; Biological; Biological Process; Biology; Birth Weight; Blood Pressure; Blood Vessels; Blood flow; Cardiac Output; Cardiovascular system; Cell model; Cells; Cyclic GMP; Cysteine; Data; Development; Endothelial Cells; Endothelium; Epidemiology; Estradiol; Estrogen Receptors; Estrogens; Event; Fetal Development; Fetal Growth; Fetal Growth Retardation; Fluorescence; Functional disorder; Funding Mechanisms; Goals; Human; In Vitro; Knowledge; Light; Link; MALDI-TOF Mass Spectrometry; Mediating; Medicine; Mitochondria; Mitochondrial Proteins; Modeling; Molecular; NG-Nitroarginine Methyl Ester; Names; Neonatal; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Organ; Outcome; Oxidation-Reduction; Pathway interactions; Perinatal; Peripheral Resistance; Phosphorylation; Physiological; Physiological Processes; Pilot Projects; Play; Post-Translational Protein Processing; Postmenopause; Pre-Eclampsia; Pregnancy; Pregnancy Complications; Production; Proteins; Proteome; Proteomics; Regulation; Research; Research Project Grants; Rest; Role; Route; Sheep; Signal Transduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Staging; Sulfhydryl Compounds; System; Testing; Time; Umbilical vein; Vasodilation; Vasodilator Agents; angiogenesis; cardiovascular disorder risk; cell motility; cofilin; fetal; gel electrophoresis; human NOS3 protein; in vivo; migration; neovascularization; novel; pregnant; protective effect; public health relevance; reproductive; response; two-dimensional

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.

描述（由申请人提供）：通过将一氧化氮（NO）衍生的亚硝酰基添加到半胱氨酸上形成S-硝基硫醇，调节大量蛋白质的功能。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与磷酸化类似。它代表了一种新兴的生理信号机制，NO 直接影响蛋白质及其功能。在这个修订后的 R21 申请中，我们的目的是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，鉴定新型内皮细胞和线粒体 SNO 蛋白，并确定 SNO-cofilin-1 的功能后遗症以及通过 eNOS 与 ER 和内源性 NO 相关的机制。目标 1：通过 CyDye Switch、2D 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) 质谱分析雌激素响应内皮细胞亚硝酰蛋白质组。目标 2：确定 SNO 蛋白响应 E22 的亚细胞定位，并通过 CyDye Switch、2D-DIGE 和 MALDI-TOF 分析内皮线粒体亚硝酰蛋白组。目标 3：确定雌激素诱导的蛋白质（例如 cofilin-1）的 S-NO 是否由特定 ER 和内源性 NO 通过 eNOS 介导的机制介导。目标 4：确定 SNO-cofilin-1 是否通过雌激素调节内皮细胞肌动蛋白重组和迁移。这项研究建立在广泛的体外和体内研究的基础上，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，拟议的研究首次迈出了下一步，以确定雌激素刺激增加 NO 产生的下游细胞和生理过程，直接影响蛋白质及其功能，因此在雌激素、NO 和内皮生物学中具有重要的生物学意义。这些研究对于围产期医学非常重要，因为内皮/NO 依赖性血管舒张是导致雌激素诱导和妊娠相关子宫血流量增加的关键机制，而子宫血流量与胎儿发育/存活和围产期/新生儿结局直接相关。这项研究还将增进我们对雌激素对心血管系统保护作用的理解。 公共健康相关性：该 R21 旨在分析内皮细胞和线粒体亚硝酰蛋白质组，希望识别新的雌激素响应亚硝酰化蛋白质靶标，并研究 cofilin-1 亚硝酰化在内皮细胞肌动蛋白组织/细胞迁移中的作用。该研究迈出了第一步，以确定雌激素刺激增加一氧化氮产生的下游事件直接影响蛋白质及其功能。获得的数据将为理解雌激素引起的和妊娠相关的子宫血管舒张提供知识飞跃，这对胎儿发育/生存和围产期/新生儿结局至关重要，也与雌激素对心血管系统的保护有关。