Troponin and myosin in regulation of muscle contraction and heart disease

肌钙蛋白和肌球蛋白调节肌肉收缩和心脏病

• 批准号: 7584532
• 负责人: Zenon Grabarek
• 金额: $ 50.75万
• 依托单位: BOSTON BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-15 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7584532
• 关键词: ATP phosphohydrolase; Actins; Actomyosin Adenosinetriphosphatase; Affect; Affinity; Binding; C-terminal; Cardiac; Cessation of life; Complement; Complex; Contractile Proteins; Data; Disease; Disulfides; Divalent Cations; Drug Design; Equilibrium; Familial Hypertrophic Cardiomyopathy; Fiber; Fluorescence Resonance Energy Transfer; Goals; Head; Heart Diseases; Heart Hypertrophy; Kinetics; Label; Lead; Measurement; Modeling; Molecular; Muscle Contraction; Muscle Fibers; Muscle function; Mutation; Myocardium; Myosin ATPase; Play; Process; Protein Isoforms; Proteins; Quality of life; Regulation; Relative (related person); Role; Signal Transduction; Skeletal Muscle; Solutions; Stretching; Structure; System; Techniques; Testing; Thin Filament; Titrations; Tropomyosin; Troponin; Troponin C; Troponin I; actin-S1; base; crosslink; genetic regulatory protein; mutant; novel; premature; protein protein interaction; public health relevance; reconstitution; reconstruction; research study; response; skeletal; tool

DESCRIPTION (provided by applicant): A number of familial hypertrophic cardiomyopathy (FHC) causing mutations have been identified in the regulatory proteins, tropomyosin (Tm) and troponin (Tn). Most of these mutations cause an increase in the Ca2+-sensitivity of muscle contraction, i.e. the onset of force occurs at lower Ca2+ concentrations. Neither the molecular mechanisms underlying the increased Ca2+-sensitivity nor its relation to the hypertrophy of the heart are well understood. Stretch activation is another cardiac phenomenon whose molecular mechanism is not understood. The long-range goal is to understand the molecular basis of FHC and stretch activation. The main hypothesis that we will test is that both of these activations involve strongly bound cross bridges (myosin heads). We plan to: 1. Determine the contribution of the myosin head-induced vs. Ca2+-induced changes in the interactions of troponin I (TnI) with actin-Tm and with troponin C (TnC) in thin filaments reconstituted with skeletal and cardiac muscle isoforms of the regulatory proteins. The main techniques will be solution ATPase and FRET measurements. 2. Determine effects of FHC mutations on occupancy of the 3 thin filament regulatory states using equilibrium titrations and transient kinetics. Fluorescent labels on selected proteins will be used to obtain equilibrium constants and rates. 3. Determine effects of selected FHC mutations in TnI and Tm on ATPase in terms of myosin vs. Ca2+ activation. FRET measurements will be used to obtain structural information. 4. Test the hypothesis that the C-terminal domain of TnC is involved in the myosin head induced activation of the thin filament. Mutants of TnC having increased affinity for Mg2+ will be used to assess the role of divalent cation in the C-domain of TnC on thin filament function. A novel mutant of TnC which reconstitutes into the thin filament and binds Ca2+ but does not activate ATPase that was developed in this lab will be used. These experiments will lead to a better understanding of the regulatory mechanism in cardiac and skeletal muscle. In particular, a better understanding of the relative contribution of the Ca2+/troponin-dependent and the myosin S1/actin-dependent activation of the thin filament will be obtained. By identifying the protein-protein interactions that are altered in the disease state it will be possible to suggest potential targets for drug design f FHC. PUBLIC HEALTH RELEVANCE Lation of muscle contraction is a complex process involving interactions among multiple proteins. As studies on familial hypertrophic cardiomyopathy (FHC) have shown even a slight change in the response of the heart muscle to the activating signal may lead to a severe disease, a diminished quality of life and premature death. The proposed studies will lead to a better understanding of the regulatory mechanism in cardiac and skeletal muscles. By identifying the aspects of the protein-protein interactions that are altered in the disease state it will be possible to suggest potential targets for drug design for FHC

描述（由申请人提供）：在调节蛋白，Tropomyosin（TM）和Troponin（TN）中已经鉴定出许多家族性肥厚性心肌病（FHC）。这些突变中的大多数导致肌肉收缩的Ca2+ - 敏感性增加，即力的发作发生在较低的Ca2+浓度下。 Ca2+敏感性增加或与心脏肥大的关系均未得到充分了解的分子机制。拉伸活化是另一种心脏现象，其分子机制尚不清楚。远程目标是了解FHC和拉伸激活的分子基础。我们将测试的主要假设是，这两种激活都涉及强绑定的跨桥（肌球蛋白头）。我们计划：1。确定肌球蛋白头引起的与Ca2+诱导的肌动蛋白I（TNI）与肌动蛋白-TM的相互作用的变化以及与调节蛋白调节蛋白的骨架和心脏肌肉同工型重构的薄丝中肌蛋白C（TNC）的贡献。主要技术将是解决方案ATPase和FRET测量。 2.使用平衡滴定和瞬态动力学来确定FHC突变对3个细丝调节状态的占用的影响。选定蛋白上的荧光标签将用于获得平衡常数和速率。 3。根据肌球蛋白与Ca2+激活，确定TNI和TM中选定的FHC突变对ATPase的影响。 FRET测量将用于获取结构信息。 4。检验以下假设：TNC的C末端结构域参与肌球蛋白头引起的细丝的激活。 TNC的突变体对Mg2+的亲和力增加将用于评估二价阳离子在TNC薄丝功能中的C域中的作用。将使用一种新型的TNC突变体，将其重构为细丝并结合Ca2+，但不会激活该实验室中开发的ATPase。这些实验将使人们对心脏和骨骼肌的调节机制有更好的了解。特别是，将获得对Ca2+/肌钙蛋白依赖性和肌动蛋白S1/肌动蛋白依赖性激活的相对贡献的更好理解。通过鉴定疾病状态中改变的蛋白质蛋白质相互作用，可以提出药物设计F FHC的潜在靶标。肌肉收缩的公共卫生相关性是一个复杂的过程，涉及多种蛋白质之间的相互作用。正如家族性心肌病（FHC）的研究表明，心肌对激活信号的反应甚至可能导致严重的疾病，生活质量降低和过早死亡。拟议的研究将使人们对心脏和骨骼肌的调节机制有更好的了解。通过识别疾病状态中改变的蛋白质蛋白质相互作用的各个方面，可以提出针对FHC的药物设计的潜在靶标