Regulation of Vascular Smooth Muscle Cytoskeleton

血管平滑肌细胞骨架的调节

• 批准号: 7742136
• 负责人: CHIH-LUEH Albert WANG
• 金额: $ 74.28万
• 依托单位: BOSTON BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 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/7742136
• 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

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（由申请人提供）：肌动蛋白细胞骨架在分化的血管平滑肌（VSM）细胞中起重要作用，在生理和病理条件下需要肌动蛋白和肌动蛋白结合蛋白（ABP）之间的密切协作。我们的总体目标是确定VSM细胞的肌动蛋白结构是通过与ABPs的相互作用来调节的机制。目前的目标是确定钙调素（CaD）和coronin的生理作用和它们之间的相互作用，在平滑肌细胞骨架的控制收缩和迁移。具体而言，我们建议：（1）验证以下假设：在VSM细胞中，CaD与coronin相互作用，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异性修饰。我们将在激动剂刺激下在A7 r5细胞和完整的小鼠VSM组织中进行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。潜在的翻译后修饰（例如，然后通过质谱法检测这些蛋白质的磷酸化）。(2)明确钙调素-coronin相互作用和磷酸化对肌动蛋白调节的结构基础。我们将测试如何磷酸化作用的钙调素表现通过相互作用，通过研究之间的结合，在肌动蛋白的存在和不存在下，使用激酶处理或磷酸化模拟蛋白质的corneumn和钙调素。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ERK外，还将检查已知磷酸化CaD和coronin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绘制出来，CaD/皮质素结合的肌动蛋白丝的结构和对丝的机械性能的影响将通过3D重建和弯曲刚度测量来确定。(3)确定钙调素-coronin相互作用的生理作用和对细胞骨架的影响。A7 r5细胞或VSM组织中的内源性CaD或coronin将首先通过siRNA处理被敲低，随后突变蛋白的再表达以测试磷酸化的调节作用。将对应于相互作用界面的合成肽作为“诱饵”引入siRNA处理的细胞。将通过检查细胞骨架结构的形态学、足状体生物发生以及迁移和收缩特性来分析这些修饰产生的影响。所有这些信息有望使我们能够机械地剖析CaD和coronin在VSM细胞中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心血管疾病，如动脉粥样硬化。420字公共卫生相关性：数百万美国人患有心血管疾病。一个主要的问题是动脉粥样硬化和再狭窄，这是由于血管平滑肌细胞的病理运动。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，我们着手研究两个关键蛋白，参与平滑肌肌动蛋白动力学和测试的假设，异常细胞外线索可以修改这些蛋白质之间的相互作用，诱导平滑肌细胞迁移。获得的信息将是非常宝贵的战斗心血管疾病仍然是头号杀手在美国。