Estrogen and Endothelial Protein Nitrosylation

雌激素和内皮蛋白亚硝基化

基本信息

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
项目类别：
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

项目摘要

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）：通过添加一氧化氮（NO）衍生的亚硝基基团在半胱氨酸中形成S-硝基硫醇，从而调节了众多蛋白质的功能。这种翻译后的蛋白质修饰被称为S-硝基化（S-NO），其生物学意义已被认为类似于磷酸化。它代表了一种新兴的生理信号传导机制，该机制无直接影响蛋白质及其功能。在此修订后的R21应用中，我们旨在鉴定新型的内皮细胞和线粒体SNO-蛋白质，并确定SNO-Cofilin-1的功能性后遗症以及与ER和ER链接的机制以及通过使用原发质的子宫动脉内皮细胞和人类Umborialical umbilicalicalicalicalicalicalicalialical静脉静脉内皮细胞相关的ER和内源性NOOS。目的1：通过Cydye开关分析雌激素反应性内皮细胞硝基蛋白蛋白质基蛋白质组，2d二维荧光差差凝胶电泳（2D-DIGE）和基质辅助激光呼吸/离子化/离子化时间（MALDI/TOF）质量图。 AIM 2：确定SNO蛋白质的亚细胞定位对E22的响应，并通过Cydye Switch，2D-DIGE和MALDI-TOF分析内皮线粒体硝基蛋白质基蛋白质组。 AIM 3：确定雌激素诱导的蛋白质（例如Cofilin-1）是否通过特定的ER和内源性NO介导的eNOS介导的ENOS介导的机制介导。目标4：确定SNO-Cofilin-1是否调节雌激素的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外和体内研究基础上，表明雌激素对内皮eNOS表达而没有产生的刺激作用。但是，拟议的研究是第一个采取下一步来确定下游细胞和生理过程的研究，这些过程通过雌激素刺激增加了无产生的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至关重要的生物学意义。这些研究在围产期医学中很重要，因为内皮/无依赖性血管舒张是导致雌激素诱导的子宫血流中与妊娠相关的关键机制，该机制与胎儿发育/生存/生存和围产期/新生儿结构直接相关。这项研究还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蛋白质体，以期识别新型的雌激素反应硝基化蛋白靶标，并研究Cofilin-1硝基化在内皮细胞肌动蛋白肌动蛋白组织/细胞迁移中的作用。该研究采取了下一步，用于确定下游事件，从而通过雌激素刺激增加一氧化氮的产生直接影响蛋白质及其功能。获得的数据将为了解雌激素引起的和妊娠相关的子宫血管舒张而提供知识飞跃，这对于胎儿发育/生存和围产期/新生儿结局至关重要，并且与心血管系统的雌激素保护有关。