Regulation of Vascular Smooth Muscle Cytoskeleton

血管平滑肌细胞骨架的调节

基本信息

批准号：
7582806
负责人：
CHIH-LUEH Albert WANG
金额：
$ 73.63万
依托单位：
BOSTON BIOMEDICAL RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-12-01 至 2012-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7582806
关键词：
Abbreviations Actin-Binding Protein Actins Actomyosin Affinity Affinity Chromatography Agonist American Architecture Atherosclerosis Binding Biochemical Biogenesis Biological Biological Assay Biological Models Blood Vessels C-terminal CSNK2A1 gene Calmodulin Calorimetry Cardiovascular Diseases Cell Line Cell Shape Cells Collaborations Complex Coupled Cues Cytoskeleton Data Disease Effectiveness Electron Microscopy Extracellular Signal Regulated Kinases Fibroblasts Filament Fluorescence Fluorescence Microscopy Fluorescence Resonance Energy Transfer Goals Health Human Immunoelectron Microscopy Immunofluorescence Immunologic Immunoprecipitation In Situ In Vitro Individual Knock-out Knockout Mice Knowledge Maps Mass Spectrum Analysis Measurement Mechanics Methods Microfilaments Microscopy Mitogen-Activated Protein Kinases Modification Molecular Molecular Weight Morphology Movement Mus Muscle Rigidity Parents Peptides Phenylephrine Phosphorylation Phosphotransferases Physiological Play Post-Translational Protein Processing Process Property Protein Kinase Protein Kinase C Proteins Proteolysis Rattus Regulation Role Sampling Site Small Interfering RNA Smooth Muscle Smooth Muscle Actin Staining Method Smooth Muscle Myocytes Structure Testing Titrations Tropomyosin Vascular Smooth Muscle Vascular Smooth Muscle Tissue Work base caldesmon casein kinase II cell motility extracellular human EMS1 protein knock-down migration mimetics mutant polymerization public health relevance reconstruction research study restenosis spatial relationship synthetic peptide

项目摘要

DESCRIPTION (provided by applicant): The actin cytoskeleton, which plays an important role in differentiated vascular smooth muscle (VSM) cells, requires close collaboration between actin and actin-binding proteins (ABPs) under physiological and pathological conditions. Our overall aim is to determine the mechanisms by which the actin architecture of VSM cells is regulated via interactions with the ABPs. The immediate goal is to define the physiological roles of caldesmon (CaD) and cortactin and their interaction in the control of smooth muscle cytoskeleton during contraction and migration. Specifically, we propose: (1) To test the hypothesis that CaD interacts with cortactin in VSM cells where the actin cytoskeleton undergoes dynamic reorganization, and that such interaction requires specific modifications on both proteins. We will carry out pull-down assays and affinity binding in A7r5 cells and intact mouse VSM tissues under agonist stimulations, and the in situ co-localization studies by immuno-microscopy. Potential post- translational modifications (e.g., phosphorylation) of these proteins will then be examined by mass spectrometry. (2) To define the structural basis of the CaD-cortactin interaction and phosphorylation on actin regulation. We will test how the phosphorylation effect on CaD is manifested through interaction with cortactin by studying the binding between cortactin and CaD in the absence and presence of actin using kinase treated or phospho-mimetic proteins. The spatial relationship in these complexes will be assessed by distance measurements and fluorescence quenching. In addition to ERK, the effect of other kinases that are known to phosphorylated CaD and cortactin will also be examined. The interaction sites will be mapped out by affinity separation coupled with proteolysis and mass spectrometric analysis, and the structure of CaD/cortactin-bound actin filaments and the effect on the mechanical properties of the filament will be determined by 3D reconstruction and flexural rigidity measurements. (3) To determine the physiological role and of the CaD-cortactin interaction and the effects on cytoskeleton. The endogenous CaD or cortactin in the A7r5 cells or VSM tissues will first be knocked down by siRNA treatment, followed by re-expression of mutant proteins to test the regulatory effect of phosphorylation. Synthetic peptides corresponding to the interaction interface will be introduced to the siRNA treated cells as "decoys". The effects resulting from these modifications will be analyzed by examining the morphology of the cytoskeleton structures, podosome biogenesis, and the migratory and contractile properties. All this information is expected to enable us to mechanistically dissect the physiological roles of CaD and cortactin in the process of the actin cytoskeleton remodeling in VSM cells, which will help us to battle cardiovascular diseases such as atherosclerosis. 420 words. PUBLIC HEALTH RELEVANCE: Millions of Americans suffer from cardiovascular diseases. A major problem is atherosclerosis and restenosis, which results from pathological movement of vascular smooth muscle cells. However, owing to the complexity of the smooth muscle cells, the molecular mechanism by which the smooth muscle cell migration is controlled remains elusive. In this project we are setting out to investigate two key proteins that are involved in the smooth muscle actin dynamics and test the hypothesis that abnormal extracellular cues can modify the interaction between these proteins and induce smooth muscle cells to migrate. Information obtained will be invaluable to battle cardiovascular diseases which remain to be number one killer in the U.S.

描述（申请人提供）：肌动蛋白细胞骨架在分化的血管平滑肌（VSM）细胞中发挥重要作用，需要肌动蛋白和肌动蛋白结合蛋白（ABP）在生理和病理条件下密切配合。我们的总体目标是确定 VSM 细胞的肌动蛋白结构通过与 ABP 相互作用进行调节的机制。近期目标是确定钙结合蛋白 (CaD) 和皮质素的生理作用以及它们在收缩和迁移过程中控制平滑肌细胞骨架中的相互作用。具体来说，我们建议：（1）检验 CaD 与 VSM 细胞中的皮质蛋白相互作用的假设，其中肌动蛋白细胞骨架经历动态重组，并且这种相互作用需要对两种蛋白质进行特定修饰。我们将在激动剂刺激下对 A7r5 细胞和完整小鼠 VSM 组织进行 Pull-down 测定和亲和结合，并通过免疫显微镜进行原位共定位研究。然后通过质谱法检查这些蛋白质的潜在翻译后修饰（例如磷酸化）。 (2) 明确CaD-cortactin相互作用和磷酸化对肌动蛋白调节的结构基础。我们将通过使用激酶处理或磷酸模拟蛋白研究在肌动蛋白不存在和存在的情况下 Cortactin 和 CaD 之间的结合，来测试 CaD 的磷酸化作用如何通过与 Cortactin 的相互作用来体现。这些复合物中的空间关系将通过距离测量和荧光猝灭来评估。除了 ERK 之外，还将检查已知磷酸化 CaD 和 cortactin 的其他激酶的作用。相互作用位点将通过亲和分离结合蛋白水解和质谱分析来绘制，CaD/cortactin 结合的肌动蛋白丝的结构以及对丝机械性能的影响将通过 3D 重建和弯曲刚度测量来确定。 (3)确定CaD-cortactin相互作用的生理作用和对细胞骨架的影响。首先通过siRNA处理敲低A7r5细胞或VSM组织中的内源性CaD或cortactin，然后重新表达突变蛋白以测试磷酸化的调节效果。对应于相互作用界面的合成肽将作为“诱饵”被引入到siRNA处理的细胞中。将通过检查细胞骨架结构的形态、足体生物发生以及迁移和收缩特性来分析这些修饰产生的效果。所有这些信息有望使我们能够从机制上剖析CaD和cortactin在VSM细胞肌动蛋白细胞骨架重塑过程中的生理作用，这将有助于我们对抗动脉粥样硬化等心血管疾病。 420字。公共卫生相关性：数百万美国人患有心血管疾病。一个主要问题是动脉粥样硬化和再狭窄，这是由血管平滑肌细胞的病理运动引起的。然而，由于平滑肌细胞的复杂性，控制平滑肌细胞迁移的分子机制仍然难以捉摸。在这个项目中，我们将着手研究参与平滑肌肌动蛋白动力学的两种关键蛋白质，并测试异常细胞外信号可以改变这些蛋白质之间的相互作用并诱导平滑肌细胞迁移的假设。所获得的信息对于对抗心血管疾病将具有无价的价值，心血管疾病仍然是美国的头号杀手。