CALDESMON--ITS ROLE IN SMOOTH MUSCLE REGULATION

Caldesmon——它在平滑肌调节中的作用

• 批准号: 6570927
• 负责人: CHIH-LUEH Albert WANG
• 金额: $ 27.63万
• 依托单位: BOSTON BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-12-01 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6570927
• 关键词: actins; calcium binding protein; caldesmon; calmodulin; chemical binding; confocal scanning microscopy; electron microscopy; enzyme activity; epitope mapping; ferrets; fluorescent dye /probe; gene expression; gene targeting; laboratory mouse; laboratory rabbit; muscle contraction; mutant; myosins; peptide chemical synthesis; phosphorylation; protein isoforms; protein localization; protein sequence; protein structure function; recombinant proteins; smooth muscle; tropomyosin; ultracentrifugation

Caldesmon (CaD) has been suggested as a thin filament-associated modulatory protein in smooth muscle cells. There has been an increasing body of evidence, based on both biochemical and cellular studies, suggesting that CaD indeed plays an inhibitory role in the actomyosin interaction, and therefore, during muscle contraction. In this proposal, efforts will be focused on the elucidation of the mechanism of smooth muscle CaD's modulatory action, and to test the hypothesis that CaD is necessary for smooth muscle to function normally. Specifically, we will: (1) Characterize the in vitro properties of unheated, native CaD and evaluate the validity of the many biochemical properties obtained with the heated, renatured CaD. (2) Test the functions of CaD with the use of fragments or antibodies. Recombinant fragments or antibodies of known epitopes of CaD will be used to test their effects on the biochemical properties of intact CaD, such as the ability to interact with calmodulin, actin, tropomyosin and myosin, and the inhibitory effect on the actomyosin ATPase activity, as well as in the native actomyosin filaments and isolated smooth muscle cells. (3) Determine the inhibitory function of an active CaD peptide, GS17C. We will use native thin filaments isolated from smooth muscle as an assay system to further test the hypothesis that GS17C exerts its antagonistic effect by binding to actin filaments and competing with the endogenous CaD in the smooth muscle cells. (4) Assess the functional roles of CaD phosphorylation and calcium binding proteins. The mechanism of reversing the CaD's inhibitory actions will be further studied in terms of the level and sites of phosphorylation of CaD and its interaction with Ca2+-binding proteins such as calmodulin and caltropin. (5) Finally, to test the postulated in vivo function of CaD by genetic approaches we will over-express CaD mutants that are either deficient in phosphorylation by MAP kinase or unable to interact with calmodulin, and seek correlation between the loss of functions and the contractile properties. These studies will generate information needed to assess the functional role of CaD in smooth muscle contraction, and afford a better understanding of the thin filament-based regulatory mechanism, which may be crucial for proper diagnosis and future development of therapeutic measures of many smooth muscle related diseases, such as gastrointestinal disorders, asthma, miscarriage and hypertension.

Caldesmon（CaD）被认为是一种与之相关的薄壳细胞， 平滑肌细胞中的调节蛋白。日益增加 基于生物化学和细胞研究的大量证据， 这表明CaD确实对肌动球蛋白起抑制作用 因此，在肌肉收缩过程中。在这项提案中， 工作重点将放在阐明平滑的机制， 肌肉钙调素的调节作用，并测试假设钙调素是 平滑肌正常发挥功能所必需的。具体而言，我们将： (1)表征未加热的天然CaD的体外性质， 评估获得的许多生化特性的有效性， 加热的，复性的CaD。(2)使用以下工具测试CaD的功能： 片段或抗体。已知抗体的重组片段或抗体 CaD的表位将被用来测试它们对生物化学的影响。 完整CaD的性质，如与钙调蛋白相互作用的能力， 肌动蛋白，原肌球蛋白和肌球蛋白，以及对肌动球蛋白的抑制作用 ATP酶活性，以及天然肌动球蛋白丝和 分离的平滑肌细胞。(3)确定一种药物的抑制功能 活性CaD肽，GS17C。我们将使用天然细丝分离， 平滑肌作为分析系统，以进一步检验GS17C 通过与肌动蛋白丝结合并竞争 与平滑肌细胞内的内源性钙调素有关。(4)评估 CaD磷酸化和钙结合蛋白的功能作用。的 对CaD抑制作用的逆转机制将作进一步的探讨 研究了CaD磷酸化的水平和位点， 与Ca2+结合蛋白如钙调素和促钙素相互作用。 (5)最后，通过遗传学方法验证CaD的体内功能。 我们将过度表达CaD突变体，这些突变体要么缺乏 通过MAP激酶磷酸化或不能与钙调蛋白相互作用，和 寻找功能丧失与收缩性 特性.这些研究将产生评估 CaD在平滑肌收缩中的功能作用，并提供更好的 对基于薄电阻的调节机制的理解， 对于正确的诊断和未来的治疗发展至关重要 许多平滑肌相关疾病的测量，如胃肠 疾病、哮喘、流产和高血压。