Deciphering how tropomyosin regulates the actin filament

破译原肌球蛋白如何调节肌动蛋白丝

• 批准号: 8040085
• 负责人: Sarah Ellen Hitchcock-DeGregori
• 金额: $ 29.64万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8040085
• 关键词: Deciphering; how; tropomyosin; regulates; actin

DESCRIPTION (provided by applicant): Regulated actin-based cellular locomotion is a property of all eukaryotic cells that is taken to the extreme of perfection in striated muscles. A small number of component parts form an assembly that responds rapidly, cooperatively and transiently to signal molecules. We have high-resolution structures of the four major components of the machine, myosin, actin, tropomyosin and troponin, but not of higher order complexes. Therefore we lack a specific mechanistic knowledge of how signaling information is communicated to and within the contractile apparatus. Mutations in the genes encoding sarcomeric proteins are the cause of cardio- and skeletal myopathies, and many of these mutations are at the ends of signaling cascades (tropomyosin and troponin-T, for example). The focus of the proposed research is one of these proteins, tropomyosin, the major regulator of the actin filament in muscle and non-muscle cells. The actin filament is the universal binding partner of tropomyosin, and there are no specific models for how or where actin binds. Our major goal is to define the molecular basis of binding specificity and regulatory function. We also present tropomyosin as a model coiled-coil, and suggest that what we learn will provide insight into how other coiled-coil proteins bind their targets and how mutations cause disease. The four aims are: Aim 1. Analysis of the molecular evolution of genes encoding tropomyosin. We will construct a phylogenetic tree, measure the rate of evolution of individual residues, and construct an ancestral tropomyosin sequence. We hypothesize that tropomyosin became "necessary" when there was a need for a more robust actin cytoskeleton than that found in amoebae. Aim 2. Structural bioinformatics analysis of phylogenetic relationships and test of hypothesis: the most conserved amino acid residues of tropomyosin include those involved in binding the highly- conserved protein actin, and regulatory functions. Conserved residues will be mutated in rat tropomyosin, and the effect on function of a recombinant protein will be tested. Aim 3. Identification of requirements for molecular recognition by tropomyosin in the actin cytoskeleton in the cellular model system, Schizosaccharomyces pombe. We will test the requirement of conserved residues of yeast tropomyosin for growth, cytoskeletal function, polarity, dynamics and formation of the contractile ring. Aim 4. Prediction of molecular recognition sites in tropomyosin and actin using molecular dynamics and docking simulations. We will construct a molecular model of predicted actin- tropomyosin complex using computational methods. PUBLIC HEALTH RELEVANCE: The main health relevance of the proposed research is to understand the molecular basis of cardio- and skeletal-myopathy-causing mutations. A bioinformatics analysis may explain why certain invertebrate tropomyosins are highly allergenic. There is the potential to develop therapeutic peptides to treat diseases involving this class of proteins.

描述（由申请人提供）：基于肌动蛋白的调节细胞运动是所有真核细胞的特性，在横纹肌中达到了极致的完美。少量的零部件形成一个组件，可以快速、协同、瞬时地响应信号分子。我们拥有机器的四个主​​要成分（肌球蛋白、肌动蛋白、原肌球蛋白和肌钙蛋白）的高分辨率结构，但没有更高阶复合物的结构。因此，我们缺乏关于信号信息如何传递到收缩装置以及在收缩装置内如何传递的具体机制知识。编码肌节蛋白的基因突变是心肌病和骨骼肌病的原因，其中许多突变发生在信号级联的末端（例如原肌球蛋白和肌钙蛋白-T）。拟议研究的重点是其中一种蛋白质，即原肌球蛋白，它是肌肉和非肌肉细胞中肌动蛋白丝的主要调节剂。肌动蛋白丝是原肌球蛋白的通用结合伴侣，对于肌动蛋白如何或在何处结合没有特定的模型。我们的主要目标是定义结合特异性和调节功能的分子基础。我们还提出了原肌球蛋白作为卷曲螺旋模型，并表明我们所学到的知识将有助于深入了解其他卷曲螺旋蛋白如何结合其靶标以及突变如何导致疾病。这四个目标是： 目标 1. 分析原肌球蛋白编码基因的分子进化。我们将构建系统发育树，测量单个残基的进化速率，并构建祖先原肌球蛋白序列。我们假设，当需要比变形虫中发现的更强大的肌动蛋白细胞骨架时，原肌球蛋白就变得“必要”。 目标2.系统发育关系的结构生物信息学分析和假设检验：原肌球蛋白最保守的氨基酸残基包括那些参与结合高度保守的肌动蛋白和调节功能的氨基酸残基。将在大鼠原肌球蛋白中突变保守残基，并测试对重组蛋白功能的影响。 目标 3. 鉴定粟酒裂殖酵母细胞模型系统中肌动蛋白细胞骨架中原肌球蛋白分子识别的要求。我们将测试酵母原肌球蛋白保守残基对生长、细胞骨架功能、极性、动力学和收缩环形成的要求。 目标 4. 使用分子动力学和对接模拟预测原肌球蛋白和肌动蛋白中的分子识别位点。我们将使用计算方法构建预测的肌动蛋白-肌球蛋白复合物的分子模型。 公共健康相关性：拟议研究的主要健康相关性是了解导致心肌病和骨骼肌病的突变的分子基础。生物信息学分析可以解释为什么某些无脊椎动物原肌球蛋白具有高度过敏性。有潜力开发治疗性肽来治疗涉及此类蛋白质的疾病。