CALDESMON--ITS ROLE IN SMOOTH MUSCLE REGULATION

Caldesmon(CAD)被认为是一种细丝相关的 平滑肌细胞中的调节蛋白。有越来越多的人 大量的证据，基于生化和细胞研究， 提示CAD确实在肌动球蛋白中起抑制作用 相互作用，因此，在肌肉收缩期间。在这份提案中， 将致力于阐明顺畅的作用机制。 肌肉CAD的调节作用，并检验CAD是 这是平滑肌肉正常运作所必需的。具体来说，我们会： (1)表征未加热、天然CAD和 评估通过以下方法获得的许多生化特性的有效性 加热、翻新的CAD。(2)利用计算机辅助设计软件进行功能测试 碎片或抗体。已知的重组片段或抗体 将利用CAD的表位来测试它们对生化的影响 完整CAD的特性，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及其对肌动球蛋白的抑制作用 ATPase活性，以及在天然肌球蛋白细丝和 分离的平滑肌细胞。(3)测定An的抑制功能。 活性CAD多肽GS17C。我们将使用天然细丝从 以平滑肌作为检测系统进一步检验GS17C的假说 通过结合肌动蛋白细丝和竞争来发挥其拮抗作用 与血管内皮细胞中的内源性CAD有关。(4)评估 CAD磷酸化和钙结合蛋白的功能作用。这个 逆转民航局抑制作用的机制将进一步 从CAD和ITS的磷酸化水平和位置方面进行研究 与钙调蛋白和钙调蛋白等钙离子结合蛋白的相互作用。 (5)从遗传学角度验证CAD的体内功能。 我们将过度表达CAD突变体的方法 被MAP激酶磷酸化或不能与钙调蛋白相互作用，以及 寻找功能丧失与收缩之间的关系 属性。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 了解基于细丝的调节机制，这可能 对于正确的诊断和未来治疗的发展至关重要 测量许多与平滑肌相关的疾病，如胃肠道疾病 疾病、哮喘、流产和高血压。

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.