CALCIUM-DEPENDENT PROTEOLYSIS IN VASCULAR SMOOTH MUSCLE

血管平滑肌中钙依赖性蛋白水解

• 批准号: 3344518
• 负责人: DAVID R HATHAWAY
• 金额: $ 10.82万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-08-01 至 1989-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3344518
• 关键词: calcium; cell growth regulation; cytoskeleton; electron microscopy; endopeptidases; enzyme structure; enzyme substrate; high performance liquid chromatography; muscle contraction; phosphorylation; proteolysis; radioimmunoassay; tissue /cell culture; vascular smooth muscle

The long-term objectives of this research proposal are two-fold: 1) to investigate the potential role of cytosolic, calcium-dependent endoproteases in the degradation of selected vascular smooth muscle cytoskeletal proteins and 2) to explore the possible role of these enxymes in disassembly of the smooth muscle cytoskeleton during the early stages of cell transformation and proliferation. We exntertain the hypothesis that calcium-dependent proteolysis is an important early step in cell proliferation and that this proteolysis is fundamental to remodeling on the vascular smooth muscle cell cytoskeleon. The specific aims that will be pursued in order to attain the long-term objectives are: 1) to purify vascular calcium-dependents proteases and characterize certain physical and structural properties, 2) to explore several avenues of potential regulation, including intramolecular and exogenous proteolysis, the presence and effect of endogenous activators and inhibitors and various forms of covalent and non-covalent protein modification, 3) to examine several purified vascular smooth muscle proteins as substrates in vitro and to determine the kinds of fragments produced, the functional activity of these fragments and what factors specifically promote or protect the selected proteins from calcium-dependent proteolysis, 4) to determine the effects of calcium-dependent proteases on contractile activity and ultrastructures of chemically-skinned vascular smooth muscle, and 5) to investigate the localization and potential function of the calcium-dependent proteases in cultured vascular smooth muscle cells treated with agents that may activate calcuim-dependent proteolysis, transform the cells or stimulate proliferation. A variety of methods will be employed including protein purification, analysis of protein structure, radioimmunoassay, measurements of muscle mechanical properties, electron microscopy and cell culture. We believe this research is important because of the link between mitogenesis and specific calcium-dependent proteases. Moreover, we believe this research pertains directly vascular smooth muscle cell proliferation which has been implicated as an early step in the disease, atherosclerosis.

这项研究建议的长远目标有两方面：1） 研究细胞溶质、钙依赖性 选择性血管平滑肌降解中的内切蛋白酶 细胞骨架蛋白和2）探索这些酶的可能作用 在平滑肌细胞骨架解体的早期阶段， 细胞转化和增殖。 我们有一个假设， 钙依赖性蛋白水解是细胞生长的重要早期步骤， 增殖，这种蛋白水解是重塑的基础上， 血管平滑肌细胞骨架。 具体目标将是 为了实现长期目标，我们所追求的是：1）净化 血管钙依赖性蛋白酶，并表征某些物理和 结构特性，2）探索潜在的几种途径 调节，包括分子内和外源性蛋白水解， 内源性激活剂和抑制剂以及各种 共价和非共价蛋白质修饰的形式，3）检查 几种纯化的血管平滑肌蛋白作为体外底物， 为了确定产生的片段的种类， 这些片段和哪些因素特别促进或保护 从钙依赖性蛋白水解中选择蛋白质，4）以确定 钙依赖性蛋白酶对收缩活性的影响， 化学皮肤血管平滑肌的超微结构，和5） 调查的定位和潜在的功能， 培养的血管平滑肌细胞中的钙依赖性蛋白酶 用可激活钙依赖性蛋白水解的试剂处理， 转化细胞或刺激增殖。 各种方法将 包括蛋白质纯化，蛋白质结构分析， 放射免疫分析，肌肉力学性能测定，电子 显微镜和细胞培养。 我们认为这项研究很重要，因为 有丝分裂和特定的钙依赖蛋白酶之间的联系。 此外，我们认为这项研究直接涉及血管平滑肌， 细胞增殖，这已经被牵连作为一个早期步骤， 疾病动脉粥样硬化