Deciphering how tropomyosin regulates the actin filament

描述（由申请人提供）：受调节的基于肌动蛋白的细胞运动是所有真核细胞的特性，在横纹肌中达到极致。少数组成部分组成一个对信号分子反应迅速、合作和短暂的组合。我们有四个主要组成部分的高分辨率结构，肌凝蛋白，肌动蛋白，原肌凝蛋白和肌钙蛋白，但没有更高阶的复合物。因此，我们缺乏一个具体的机制知识的信号信息是如何沟通和内部的收缩装置。编码肌合成蛋白的基因突变是导致心肌病和骨骼肌病的原因，其中许多突变位于信号级联的末端（例如原肌球蛋白和肌钙蛋白- t）。拟议研究的重点是这些蛋白质中的一种，原肌球蛋白，肌肉和非肌肉细胞中肌动蛋白丝的主要调节因子。肌动蛋白丝是原肌凝蛋白的普遍结合伙伴，肌动蛋白如何结合或在哪里结合尚无具体模型。我们的主要目标是确定结合特异性和调节功能的分子基础。我们还将原肌球蛋白作为一种螺旋状蛋白模型，并建议我们所了解的将为其他螺旋状蛋白如何结合其靶标以及突变如何引起疾病提供见解。这四个目标是：原肌球蛋白编码基因的分子进化分析。我们将构建一个系统发育树，测量单个残基的进化速率，并构建一个祖先原肌球蛋白序列。我们假设，当需要比在变形虫中发现的更强健的肌动蛋白细胞骨架时，原肌凝蛋白成为“必要的”。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与高度保守的肌动蛋白结合的氨基酸残基和调节功能氨基酸残基。将原肌球蛋白的保守残基进行突变，并测试其对重组蛋白功能的影响。目标3。裂糖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需求的鉴定。我们将测试酵母原肌球蛋白的保守残基对生长、细胞骨架功能、极性、动力学和收缩环形成的要求。目标4。利用分子动力学和对接模拟预测原肌凝蛋白和肌动蛋白的分子识别位点。我们将用计算方法构建预测肌动蛋白-原肌球蛋白复合物的分子模型。

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.