Deciphering how tropomyosin regulates the actin filament

描述（由申请人提供）：基于肌动蛋白的细胞运动是所有真核细胞的一种特性，在横纹肌中达到了极致。少量的组成部分形成一个组件，快速，合作和短暂的信号分子的反应。我们已经有了这个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，但没有更高级的复合物。因此，我们缺乏一个具体的机制知识，信号信息是如何沟通，并在收缩装置。编码肌节蛋白的基因突变是心脏和骨骼肌病变的原因，并且这些突变中的许多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之一，原肌球蛋白，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，并且对于肌动蛋白如何或在何处结合没有特定的模型。我们的主要目标是确定结合特异性和调节功能的分子基础。我们还提出原肌球蛋白作为模型卷曲螺旋，并建议我们所了解的将提供洞察其他卷曲螺旋蛋白如何结合其目标和突变如何导致疾病。这四个目标是：目标1。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，测量个别残基的进化速率，并构建一个祖先原肌球蛋白序列。我们假设，原肌球蛋白成为“必要的”时，有需要一个更强大的肌动蛋白细胞骨架比阿米巴。 目标二。结构生物信息学分析系统发育关系和假设检验：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蛋白肌动蛋白，以及调节功能的残基。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：在粟酒裂殖酵母的细胞模型系统中，原肌球蛋白在肌动蛋白细胞骨架中分子识别的要求的鉴定。我们将测试酵母原肌球蛋白的保守残基的生长，细胞骨架功能，极性，动力学和收缩环的形成的要求。 目标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。我们将用计算方法构建一个预测的肌动蛋白-原肌球蛋白复合物的分子模型。 公共卫生相关性：拟议研究的主要健康相关性是了解引起心脏和骨骼肌病变突变的分子基础。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是高度过敏原。存在开发治疗肽以治疗涉及这类蛋白质的疾病的潜力。

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.