Deciphering how tropomyosin regulates the actin filament

描述(由申请人提供)：受调控的基于肌动蛋白的细胞运动是所有真核细胞的特性，在横纹肌中达到了极致。少量的组成部件组成一个组件，对信号分子做出快速、协作和瞬时的反应。我们有机器的四个主要组成部分的高分辨率结构，肌球蛋白、肌动蛋白、原肌球蛋白和肌钙蛋白，但没有更高阶的复合体。因此，我们缺乏关于信号信息如何传递到收缩装置和收缩装置内的具体机械性知识。肌节蛋白编码基因的突变是心脏和骨骼肌病的原因，其中许多突变位于信号级联反应的末端(例如，原肌球蛋白和肌钙蛋白-T)。拟议的研究重点是这些蛋白质之一，原肌球蛋白，肌肉和非肌肉细胞中肌动蛋白细丝的主要调节因子。肌动蛋白细丝是原肌球蛋白的通用结合伙伴，目前还没有关于肌动蛋白如何或在哪里结合的特定模型。我们的主要目标是确定结合的特异性和调节功能的分子基础。我们还介绍了原肌球蛋白作为卷曲螺旋的模型，并建议我们所了解的将为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提供洞察力。目的：1.原肌球蛋白编码基因的分子进化分析。我们将构建系统发育树，测量个体残基的进化速度，并构建祖先原肌球蛋白序列。我们假设，当需要比阿米巴中发现的更强健的肌动蛋白细胞骨架时，原肌球蛋白变得“必要”。目的2.系统发育关系的结构生物信息学分析和假说的检验：原肌球蛋白最保守的氨基酸残基包括与高度保守的蛋白质肌动蛋白结合的氨基酸残基和调节功能。保守残基将在大鼠原肌球蛋白中发生突变，并将测试重组蛋白对功能的影响。目的3.在裂殖酵母细胞模型系统中鉴定原肌球蛋白对肌动蛋白细胞骨架分子识别的要求。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需求。目的4.用分子动力学和对接模拟预测原肌球蛋白和肌动蛋白的分子识别位点。我们将使用计算方法构建预测的肌动蛋白-原肌球蛋白复合体的分子模型。 公共卫生相关性：拟议研究的主要健康相关性是了解导致心脏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。有可能开发治疗性多肽来治疗涉及这类蛋白质的疾病。

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.