THIN FILAMENTS AND MUSCLE REGULATION

细丝和肌肉调节

• 批准号: 2609240
• 负责人: WILLIAM J LEHMAN
• 金额: $ 25万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-30 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2609240
• 关键词: Rana; actins; caldesmon; calponin; electron microscopy; image processing; microfilaments; muscle contraction; muscle proteins; myosins; protein reconstitution; protein structure; smooth muscle; striated muscles; tropomyosin; troponin

Thin filament-associated regulatory proteins control actomyosin interactions in a variety of muscle and non-muscle contractile systems. In vertebrate striated muscle, the regulatory protein complex of tropomyosin and troponin linked to actin in the thin filaments causes relaxation by blocking strong myosin-crossbridge binding onto actin in the absence of Ca2+. In smooth muscle, thin filament-associated proteins (tropomyosin, caldesmon and possibly calponin) may function in conjunction with or in addition to the well-known Ca2+-calmodulin- dependent myosin phosphorylation process to modulate actomyosin ATPase and consequently tension generation and/or maintenance. In non-muscle systems, caldesmon linked to actin may, in concert with myosin phosphorylation, also act to regulate actomyosin-dependent cytoplasmic motility. The caldesmon-tropomyosin complex, like troponin-tropomyosin, inhibits actomyosin ATPase at low intracellular Ca2+-concentration, while tropomyosin itself potentiates ATPase. We will investigate the molecular mechanisms by which thin filament-linked proteins influence actomyosin by studying The structural interactions of the proteins on thin filaments from different types of muscles and other cells. Electron microscopy (including cryomicroscopy), computer-assisted image analysis and three- dimensional reconstruction will be used to determine thin filament structure and to evaluate changes in the structural arrangement of thin filament components in "on-" and in "off-states". Reconstruction of troponin- and caidesmon-based native thin filaments as well as reconstruction of synthetic filaments reconstituted from the components of these systems will be carried out to determine the impact of troponin, caldesmon and calponin on tropomyosin position, two-domain actin structure, and on actomyosin-binding. Reconstructions of high precision will be fitted to the atomic map of F-actin to detail specific atomic contacts between regulatory proteins and functional domains on F-actin. Our own published reconstructions and those of others demonstrate the feasibility of these goals. We anticipate that our continued structural studies will lead to an elucidation of the molecular mechanism of troponin action in skeletal muscle and contribute towards an understanding of the role and mechanism of caldesmon and calponin in the fine tuning of the contractile response in smooth muscle. A broad understanding of the molecular mechanisms involved in the regulation of contractility in healthy tissue may aid in future evaluation of defects occurring in some disease procosses. The control of smooth muscle contractility, for example, is of great importance in the regulation of vascular tone and pulmonary airway resistance, determinants in a number of conditions such as hypertension and asthma. Moreover, the general significance of our goals is underscored by the possibility that caldesmon linked to non-muscle microfilaments may have a role in controlling cytoplasmic motile processes such as cytokinesis, and therefore may be involved in regulating cell division in normal and cancer cells.

细丝相关调节蛋白控制肌动球蛋白 各种肌肉和非肌肉收缩系统中的相互作用。 在脊椎动物横纹肌中，调节蛋白复合物 原肌球蛋白和肌钙蛋白与细丝中的肌动蛋白相连，导致 通过阻断与肌动蛋白的强肌球蛋白跨桥结合来放松 缺乏Ca2+。在平滑肌中，细丝相关蛋白 （原肌球蛋白、钙结合蛋白和可能的钙调蛋白）可能在 与众所周知的 Ca2+-钙调蛋白-结合或补充 依赖肌球蛋白磷酸化过程来调节肌动球蛋白 ATP 酶 以及因此产生和/或维持张力。在非肌肉 系统中，与肌动蛋白相连的卡尔德斯蒙可能与肌球蛋白协同作用 磷酸化，还可以调节肌动球蛋白依赖性细胞质 动力。 钙结合蛋白-原肌球蛋白复合物，如肌钙蛋白-原肌球蛋白， 在低细胞内 Ca2+ 浓度下抑制肌动球蛋白 ATP 酶，同时 原肌球蛋白本身可增强 ATP 酶。我们将研究分子 细丝连接蛋白影响肌动球蛋白的机制 通过研究细丝上蛋白质的结构相互作用 来自不同类型的肌肉和其他细胞。电子显微镜 （包括冷冻显微镜）、计算机辅助图像分析和三 维度重建将用于确定细丝 结构并评估薄层结构排列的变化 灯丝组件处于“开”和“关”状态。重建 基于肌钙蛋白和 caidesmon 的天然细丝以及 由成分重构的合成长丝的重建 这些系统将被用来确定肌钙蛋白的影响， 原肌球蛋白位置上的钙结合蛋白和钙调蛋白，二域肌动蛋白 结构和肌动球蛋白结合。高精度重建 将拟合至 F-肌动蛋白的原子图以详细描述特定原子 调节蛋白和 F-肌动蛋白功能域之间的接触。 我们自己发表的重建和其他人的重建证明了 这些目标的可行性。我们预计我们的持续结构性 研究将有助于阐明其分子机制 肌钙蛋白在骨骼肌中的作用并有助于 了解caldesmon和calponin在体内的作用和机制 微调平滑肌的收缩反应。广泛的 了解参与调节的分子机制 健康组织的收缩性可能有助于未来评估缺陷 发生在某些疾病过程中。平滑肌的控制 例如，收缩性对于调节 血管张力和肺气道阻力，许多因素的决定因素 高血压和哮喘等疾病。 此外，一般 我们的目标的重要性通过以下可能性得到强调： 与非肌肉微丝相连的卡尔德斯蒙可能在 控制细胞质运动过程，例如胞质分裂，以及 因此可能参与正常细胞分裂的调节 癌细胞。