Regulation of the Smooth Muscle Cytoskeleton

平滑肌细胞骨架的调节

• 批准号: 7329701
• 负责人: CHIH-LUEH Albert WANG
• 金额: $ 47.23万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7329701
• 关键词: Actin-Binding Protein; Actins; Affect; Affinity; Binding; Binding Sites; Biological Assay; Blood Vessels; Calorimetry; Cardiovascular Diseases; Cell Shape; Cells; Column Chromatography; Complement; Coupled; Cysteine; Cytoskeleton; Data; Disease; Elements; Engineering; Exhibits; F-Actin; Fluorescence; Fluorescence Resonance Energy Transfer; Genes; Hypertension; Immunoelectron Microscopy; In Vitro; Methods; Modeling; Molecular; Molecular Conformation; Monitor; Morphology; Numbers; Parents; Peptide Fragments; Peptides; Phosphorylation; Phosphorylation Site; Physiological; Play; Process; Property; Protein Conformation; Proteins; Proteolysis; Reagent; Recombinants; Regulation; Research Personnel; Role; Sedimentation process; Shapes; Signal Transduction; Site; Smooth Muscle; Smooth Muscle Myocytes; Structure; Surface Plasmon Resonance; Testing; Therapeutic; Titrations; Vascular Smooth Muscle; Work; ZYX gene; base; blood pressure regulation; caldesmon; calponin; design; human EMS1 protein; in vivo; insight; mouse model; mutant; novel; research study; synthetic peptide; tool; vasodilator-stimulated phosphoprotein

Actin cytoskeleton plays an important role in differentiated vascular smooth muscle cells (dVSMCs) because they maintain and change the cell shape in concert with the activities of the contractile apparatus. A large number of actin-binding proteins (ABPs) participate in these processes, many of which contain multiple binding modules and are subject to phosphorylation. The underlying hypothesis of Project 2 is that phosphorylation alters the actin-binding properties of ABPs as a consequence of cell signaling, and thereby allows for remodeling of the actin cytoskeleton. This hypothesis will be tested the mechanistic basis for such remodeling and its regulation in dVSMCs will be investigated. With previously demonstrated approaches, the phosphorylation-dependent conformational changes will be determined using five selected cytoskeleton proteins (caldesmon, calponin, cortactin, VASP and zyxin) as model cases. The structural information thus obtained will be compared to the results from Projects 3 and 4. A second hypothesis that cytoskeleton proteins work together as partners to synergistically maintain the actin cytoskeleton structures will also be tested. In support of this idea a recently developed mouse model in which the smooth muscle caldesmon gene is disrupted, demonstrated that a number of cytoskeleton proteins exhibited decreased levels of expression along with the elimination of smooth muscle caldesmon. The potential interactions between caldesmon and these proteins will be examined. Novel biophysical tools such as surface plasmon resonance and isothermal titration calorimetry will be used. In addition, the sites of interaction will be determined and synthetic peptides or recombinant fragments will be prepared corresponding to the binding sequences. These peptides/fragments will then be used as decoys to test the functional significance of the interactions and phosphorylation in primary SMCs and in isolated dVSMCs. The localization of cytoskeleton proteins as well as the morphology of the cytoskeleton structure will be examined by immuno-fluorescence and immuno-electron microscopy, together with the findings from Project 1, to gain insights into the effects on smooth muscle contractility. Since the contraction of vascular smooth muscles controls the blood pressure in our body, mulfunction of vascular smooth muscle leads to hypertension and other cardiovascular diseases. Information obtained from this study will help develop therapeutic reagents for these diseases.

肌动蛋白细胞骨架在分化的血管平滑肌细胞中起着重要作用，因为 它们与收缩机构的活动相一致地维持和改变细胞的形状。大号 许多肌动蛋白结合蛋白(ABPs)参与这些过程，其中许多包含多个 结合模块，并受制于磷酸化。项目2的基本假设是 作为细胞信号传递的结果，磷酸化改变了ABPs的肌动蛋白结合特性，从而 允许肌动蛋白细胞骨架的重塑。这一假设将被检验为这样的机制基础 将对dVSMCs的重塑及其调控进行研究。使用先前演示的方法， 依赖于磷酸化的构象变化将使用五个选定的细胞骨架来确定 蛋白质(钙调蛋白、钙蛋白、皮质素、血管活性蛋白和凝血酶)作为模型病例。因此，结构信息 将得到的结果与项目3和项目4的结果进行比较。第二个假设是细胞骨架 蛋白质作为合作伙伴协同工作，以协同维持肌动蛋白的细胞骨架结构 测试过。为了支持这一观点，最近开发了一种小鼠模型，在该模型中，平滑肌钙调蛋白 基因被破坏，表明一些细胞骨架蛋白显示出降低的水平 表达伴随着平滑肌钙调蛋白的消除。两国之间的潜在互动 钙离子和这些蛋白质将被检测。新型生物物理工具，如表面等离子激元 将使用共振法和等温滴定量热法。此外，互动的网站将是 将根据结合情况制备相应的确定和合成的多肽或重组片段 序列。然后，这些多肽/片段将被用作诱饵，以测试 原代SMC和分离的dVSMC的相互作用和磷酸化。细胞骨架的定位 蛋白质和细胞骨架结构的形态将通过免疫荧光进行检测。 和免疫电子显微镜，结合项目1的发现，以获得对影响的洞察 关于平滑肌肉的收缩能力。因为血管平滑肌的收缩控制着血液 我们体内的压力，血管平滑肌的多功能导致高血压和其他心血管疾病 疾病。从这项研究中获得的信息将有助于开发这些疾病的治疗试剂。