Estrogen and Endothelial Protein Nitrosylation

雌激素和内皮蛋白亚硝基化

• 批准号: 8119098
• 负责人: DONGBAO CHEN
• 金额: $ 19.13万
• 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 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8119098
• 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; Cell physiology; 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; Protein S; Proteins; Proteome; Proteomics; Regulation; Research; Research Project Grants; Rest; Role; Route; SKIL gene; 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(申请人提供)：通过将一氧化氮(NO)衍生的亚硝基加到半胱氨酸上形成S-亚硝硫醇，调节过多蛋白质的功能。这种翻译后的蛋白质修饰被命名为S亚硝化(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，它直接影响蛋白质及其功能。在这次修订的R21应用中，我们的目的是鉴定新的内皮细胞和线粒体SNO-蛋白，并以原代子宫动脉内皮细胞和人脐静脉内皮细胞为模型，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。目的：应用CyDye Switch、2D-DIGE和基质辅助激光解吸电离飞行时间质谱仪(MALDI/TOF)分析雌激素反应的内皮细胞亚硝基蛋白质组。目的：利用CyDye Switch、2D-DGE和MALDI-TOF分析内皮细胞线粒体亚硝基蛋白质组，确定E22对SNO-蛋白的亚细胞定位。目的：探讨雌激素是否通过内皮型一氧化氮合酶(ENOS)介导的特异性ER和内源性NO(S)介导COFILIN-1等蛋白的表达。目的4：确定SNO-cofilin-1是否通过雌激素调节内皮细胞肌动蛋白的重组和迁移。这项研究建立在广泛的体内外研究的基础上，表明雌激素对内皮eNOS表达和NO产生的刺激作用；然而，本研究首次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关键的生物学意义。这些研究在围产期医学中很重要，因为内皮/一氧化氮依赖性血管扩张是雌激素诱导的和妊娠相关的子宫血流量增加的关键机制，直接与胎儿发育/存活和围产儿/新生儿结局相关。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：本R21旨在分析内皮细胞和线粒体亚硝基蛋白质组，以期识别新的雌激素反应亚硝酸化蛋白靶标，并研究cofilin-1亚硝化在内皮细胞肌动蛋白组织/细胞迁移中的作用。这项研究迈出了下一步的第一步，以确定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。所获得的数据将为理解雌激素诱导和妊娠相关的子宫血管扩张提供知识飞跃，这些血管扩张对胎儿发育/存活和围产期/新生儿结局至关重要，也与雌激素对心血管系统的保护有关。