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 (dVSMC) 中发挥重要作用，因为 它们与收缩装置的活动一致地维持和改变细胞形状。一个大 许多肌动蛋白结合蛋白（ABP）参与这些过程，其中许多包含多个 结合模块并进行磷酸化。项目 2 的基本假设是 由于细胞信号传导，磷酸化改变了 ABP 的肌动蛋白结合特性，从而 允许重构肌动蛋白细胞骨架。这一假设将被检验其机制基础 将研究 dVSMC 中的重塑及其调节。通过先前演示的方法， 将使用五种选定的细胞骨架来确定磷酸化依赖性构象变化 蛋白质（caldesmon、calponin、cortactin、VASP 和 zyxin）作为模型病例。因此结构信息 获得的结果将与项目 3 和 4 的结果进行比较。第二个假设是细胞骨架 蛋白质作为伙伴一起工作，协同维持肌动蛋白细胞骨架结构也将 已测试。为了支持这一想法，最近开发了一种小鼠模型，其中平滑肌钙质蛋白 基因被破坏，证明许多细胞骨架蛋白的水平下降 随着平滑肌钙蛋白的消除而表达。之间潜在的相互作用 caldesmon 和这些蛋白质将被检查。新型生物物理工具，例如表面等离子体 将使用共振和等温滴定量热法。此外，互动地点将是 确定并制备与结合相对应的合成肽或重组片段 序列。然后，这些肽/片段将被用作诱饵来测试该肽/片段的功能意义。 原代 SMC 和分离的 dVSMC 中的相互作用和磷酸化。细胞骨架的定位 蛋白质以及细胞骨架结构的形态将通过免疫荧光检查 和免疫电子显微镜，以及项目 1 的研究结果，以深入了解效果 关于平滑肌收缩力。由于血管平滑肌的收缩控制着血液 体内压力过大，血管平滑肌功能失调，导致高血压等心血管疾病 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。