Thin Filaments and Muscle Regulation

细丝和肌肉调节

• 批准号: 10355843
• 负责人: WILLIAM J LEHMAN
• 金额: $ 41.25万
• 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 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10355843
• 关键词: Actin-Binding Protein; Actins; Actomyosin; Address; Affect; Binding; Biochemical; Biochemistry; Biological Assay; Biomedical Research; Boston; C-terminal; Cardiac; Cardiac Muscle Contraction; Cardiomyopathies; Clinical; Communication; Complex; Computer Models; Coupled; Cryoelectron Microscopy; Data; Defect; Disease; Disease Outcome; Disease Progression; Docking; Face; Filament; Functional disorder; Genes; Genetic; Goals; Head; Heart; Homeostasis; Hot Spot; In Vitro; Intervention; Lead; Left; Link; Location; Measures; Microfilaments; Modeling; Molecular; Molecular Conformation; Muscle; Muscle Contraction; Muscle Proteins; Muscle relaxation phase; Mutation; Myocardium; Myopathy; Myosin ATPase; Pathologic; Pathway interactions; Penetrance; Phenotype; Physiological; Physiology; Plant Roots; Play; Positioning Attribute; Process; Proteins; Protocols documentation; Regulation; Regulatory Pathway; Relaxation; Research; Research Personnel; Role; Severities; Signal Pathway; Site; Skeletal Muscle; Specific qualifier value; Structure; Symptoms; Tail; Testing; Thin Filament; Three-Dimensional Image; Tissue Model; Tissues; Translations; Tropomyosin; Troponin; Universities; Work; X-Ray Crystallography; base; cardiac tissue engineering; cell motility; computerized tools; defined contribution; design; drug development; druggable target; genetic regulatory protein; image reconstruction; insight; link protein; molecular dynamics; mutant; predictive test; reconstruction; response; small molecule; synergism; tool

Thin filament-linked actin-binding proteins, troponin and tropomyosin, control actomyosin-based muscle contraction in cardiac and skeletal muscles. To elucidate mechanisms of muscle thin filament function at a fundamental molecular level, it is crucial to determine the changing structural interactions of these regulatory proteins that control muscle cooperative activation and relaxation via allosteric communication pathways between filament components. It follows that disease-related myofibrillar protein mutants can perturb muscle on-off switching by causing an imbalance in troponin-tropomyosin interactions on actin which, in turn, destabilizes relaxed or active states and the transitions between them. It is our premise that early stage intervention to correct such imbalances is paramount in diminishing or reversing resulting inexorable disease progression. In the current work, we will address these imbalances by taking a multifaceted structural approach to elucidate the mechanism of thin filament regulation and thus establish root causes of these perturbations. To accomplish this goal: 1. We will use cryo-electron microscopy, coupled with 3D-image reconstruction, to establish regulatory transitions of troponin and tropomyosin as well as test the impact of myosin-binding on thin filament actin and tropomyosin. 2. We will refine this experimental approach with computational tools that we have pioneered to bring cryo-EM structures even closer to an atomic level using protein-protein docking protocols and molecular dynamics. 3. We will compare structural interactions that occur in normal thin filaments with those in filaments containing mutant proteins linked to myopathies in order to assess how mutation-linked aberrant physiology can link to myopathology, while localizing druggable target pockets at protein-protein interfaces. To achieve our aims, (1) we will focus on identifying structural domains at the interface between of troponin subunit-T and actin-tropomyosin (Specific Aim 1); (2) we will reveal the structural mechanism used by regulatory domains of troponin subunit-I to trap tropomyosin in its relaxed-state position on actin (Specific Aim 2); (3) we will determine the impact of myosin structural interactions on actin-tropomyosin, less recognized but significant effectors of thin filament regulation (Specific Aim 3). The influence of myopathic-linked mutations in troponin, tropomyosin, actin and myosin will not only be predicted and tested structurally but assayed functionally by measuring in vitro motility and contractility in engineered heart tissue. Aiming to develop tools to counteract regulatory imbalances, we collaborate with associates at the Boston University Central for Molecular Discovery to identify small molecules to be trapped at druggable interfaces along thin filaments in order to potentially manipulate cooperative, regulatory pathways. Thus, our work on the molecular regulation of cardiac and skeletal muscle thin filaments and muscle contraction lies at the intersection of basic and translation biomedical research. Here, our overarching goal is to couple understanding of atomic level mutational “insults” that alter muscle control mechanisms to prospects of reversing early-stage defects in physiological function.

肌钙蛋白和原肌球蛋白的细丝连接肌动蛋白结合蛋白控制肌动球蛋白 心肌和骨骼肌的收缩。为了阐明肌肉细丝功能的机制 在基础分子水平上，确定这些调节分子的结构相互作用的变化是至关重要的 通过变构通讯途径控制肌肉协同激活和松弛的蛋白质 灯丝组件。因此，与疾病相关的肌原纤维蛋白突变体可以扰乱肌肉的开关。 肌钙蛋白-原肌球蛋白相互作用的不平衡导致肌动蛋白的转换，进而破坏松弛的稳定性 或活动状态以及它们之间的转换。我们的前提是，早期干预以纠正这种情况 在减少或逆转导致不可避免的疾病发展方面，失衡是至关重要的。在目前的工作中， 我们将通过采取多方面的结构方法来解决这些失衡问题，以阐明Thin的机制 细丝调节，从而确定这些扰动的根本原因。要实现这一目标：1.我们将使用 冷冻电子显微镜，结合3D图像重建，以建立肌钙蛋白和 并检测肌球蛋白结合对细丝肌动蛋白和原肌球蛋白的影响。2.我们将精细化 这一实验方法使用了我们首创的计算工具，使低温电磁结构 使用蛋白质-蛋白质对接协议和分子动力学更接近原子水平。3.我们会比较 正常细丝与含有连锁突变蛋白的细丝发生的结构相互作用 以评估突变相关的异常生理学如何与肌肉病理学相联系，而 在蛋白质-蛋白质交界处定位可用药靶区。为实现我们的目标，(1)我们将重点抓好 确定肌钙蛋白亚基-T和肌动蛋白原肌球蛋白界面的结构域(特定目的 1)；(2)我们将揭示肌钙蛋白亚基-I调节域捕获原肌球蛋白的结构机制 在其松弛状态下对肌动蛋白的影响(特定目标2)；(3)我们将确定肌球蛋白结构的影响 肌动蛋白-原肌球蛋白的相互作用，较少被认识但却是细丝调节的重要效应物(特定目的 3)。肌钙蛋白、原肌球蛋白、肌动蛋白和肌球蛋白的肌病相关突变的影响不仅可以预测。 并通过测量工程心脏的体外运动和收缩能力来进行结构测试和功能测试 组织。为了开发工具来抵消监管失衡，我们与波士顿的同事们合作 分子发现大学中心识别被困在可药物界面的小分子 细丝，以便潜在地操纵合作的、调节的通路。因此，我们在分子方面的工作 心肌和骨骼肌细丝和肌肉收缩的调节处于基础和 翻译生物医学研究。在这里，我们的首要目标是结合对原子水平突变的理解 改变肌肉控制机制的“侮辱”，对扭转生理功能早期缺陷的前景。