Thin Filaments and Muscle Regulation

描述(申请人提供)：细丝相关肌动蛋白结合蛋白控制肌肉和非肌肉细胞中基于肌球蛋白的收缩和细胞骨架的形成。要阐明这些机制，关键是要确定所涉及的蛋白质的结构相互作用。我们讨论了几个与了解肌肉细丝功能相关的基本问题：(1)骨骼肌、心肌和平滑肌中细丝的结构是什么？(2)调控肌肉活动的细丝连接蛋白的结构相互作用是什么？(3)这些蛋白如何与肌动蛋白相互作用形成肌肉细胞骨架？我们使用最先进的电子显微镜、图像分析和三维重建来建立结合在细丝肌动蛋白上的成分的大分子结构。使用这些技术：(A)我们的目的是通过分析原肌球蛋白和肌钙蛋白对细丝的相互作用以及钙离子和肌球蛋白交叉桥结合的影响来确定肌钙蛋白原肌球蛋白调节骨骼肌和心肌活动的结构基础。(B)为了帮助了解肌肉细胞的皮质肌动蛋白细胞骨架的组织，我们旨在通过检测它们不同的钙蛋白同源(CH)结构域的结合来确定dystrophin和utroin与丝状F-肌动蛋白的结构相互作用。(C)我们的目标是评估细丝相关钙调蛋白作为基于肌动球蛋白的运动和细胞骨架组装在平滑肌中的可能的调节器的结构作用。(D)对与肌动蛋白结合的星云蛋白结构的研究将是我们继续研究细丝功能设计的一部分。在每项研究中，重构都将与F-肌动蛋白的原子分辨图谱相适应，以确定结合蛋白与肌动蛋白的分子接触。此外，这种“混合结晶学”将用于在EM密度图中拟合新解决的肌钙蛋白、原肌球蛋白、肌营养不良蛋白和中性粒细胞营养素结构域的晶体结构，以达到接近原子的分辨率。我们正在进行的肌钙蛋白-海藻蛋白调节肌丝的研究将有助于阐明骨骼肌和心肌松弛和激活的分子机制。我们成功启动的关于中性粒细胞营养素和营养不良蛋白的结构研究将确立它们作为细胞骨架元素的独特特征，这些信息适用于设计肌肉营养不良的基因疗法。对平滑肌细丝的研究将有助于理解平滑肌收缩反应的微调。这种调节影响血管张力和肺气道阻力，这是高血压和哮喘等疾病的决定因素。肌动蛋白和相关蛋白在多种重要的细胞机制中的作用突显了我们目标的更广泛意义，这些机制可能会在恶性肿瘤中变得异常。

DESCRIPTION (provided by applicant): Thin filament-associated actin-binding proteins control both actomyosin-based contractility and cytoskeletal formation in muscle and non-muscle cells. To elucidate these mechanisms, it is crucial to determine the structural interactions of the proteins involved. We address several interrelated questions fundamental to understanding muscle thin filament function: (1) What is the architecture of the thin filament in skeletal, cardiac and smooth muscles? (2) What are the changing structural interactions of thin filament-linked proteins that regulate muscle activity? (3) How do these proteins interact with actin to form the muscle cytoskeleton? We use state-of-the-art electron microscopy, image analysis and 3-D reconstruction to establish he macromolecular structure of components bound on thin filament actin. Using these techniques: (A) We aim to determine the structural basis of troponin-tropomyosin regulation of skeletal and cardiac muscle activity by analyzing interactions of tropomyosin and troponin on thin filaments and the effects of Ca2+ and myosin-crossbridge binding. (B) To help understand the organization of the cortical actin cytoskeleton of muscle cells, we aim to determine the structural interactions of dystrophin and utrophin with filamentous F-actin by examining the binding of their distinct calponin homology (CH)-domains. (C) We aim to assess the structural role of thin filament associated caldesmon as a possible modulator of actomyosin-based motility and cytoskeletal assembly in smooth muscle. (D) Studies of the structure of nebulin bound to actin will be part of our continuing investigation of the functional design of thin filaments. In each study, reconstructions will be fitted to the atomic resolution maps of F-actin to define molecular contacts of binding proteins with actin. Further, such "hybrid crystallography" will be used to fit newly solved crystal structures of troponin, tropomyosin, dystrophin and utrophin domains within EM density maps to attain near atomic resolution. Our ongoing studies on troponin-trepomyosin regulated filaments will lead to an elucidation of the molecular mechanism of relaxation and activation in skeletal and cardiac muscle. Our successfully initiated structural studies on utrophin and dystrophin will establish their unique features as cytoskeletal elements, information applicable to designing genetic therapies for muscular dystrophy. Studies on smooth muscle filaments will contribute to understanding the fine-tuning of the smooth muscle contractile response. Such modulation affects vascular tone and pulmonary airway resistance, determinants in, e.g., hypertension and asthma. The wider significance of our goals is underscored by the role of actin and associated proteins in diverse and vital cellular mechanisms that can become aberrant in malignancy.