Factors influencing regulation of the dynamics of the actin filament pointed end

描述(由申请人提供)：肌动蛋白末端动力学的调节在横纹肌肌节以及平滑肌和非肌肉细胞中的细丝组装中至关重要。肌动蛋白细丝有两个截然不同的末端：快速生长的带刺末端和生长较慢的尖端。原肌球蛋白(Tmod)是一种与原肌球蛋白(TM)结合的蛋白，它结合并调节尖端的动力学。这项提议的重点是定义Tmod的结构-功能关系，以及与其功能结合并调节其功能的蛋白质。在许多可能影响肌动蛋白末端动力学的可能机制中，似乎最有可能的三种机制将被探索：Tmod/TM相互作用中的异构体依赖(目标1)，识别改变Tmod功能的未知Tmod结合伙伴(目标2)和Tmod磷酸化(目标3)。在体外实验中，肌动蛋白的封顶是紧密的；然而，在活的心肌细胞中，封顶是短暂的。必须有一种机制来调节Tmod，要么是通过结合Tmod来调节的蛋白质，要么是通过共价修饰来调节的蛋白质。在目标1中，将使用TM N端与Tmod结合的模型肽以及Tmod上两个已定义的TM结合位点的模型肽来了解不同TM与不同Tmod结合的特异性的结构基础。结合将使用天然凝胶电泳法、交联法和圆二色谱进行分析。然后，将在尖端延长试验和使用Tmod1缺失的胚胎干细胞的抢救实验中评估这些特异性。目的2验证假设：Tmod中的富含亮氨酸重复序列(LRR)结构域与调节其功能的因子结合。来自骨骼肌的不纯肌动蛋白制剂含有一种负面调节Tmod封顶的因子。这种活性取决于LRR结构域的存在，这是一个通常参与蛋白质-蛋白质相互作用的基序。其目的将是提纯、识别和表征调控因素。目的3研究TRPM7激酶对Tmod的磷酸化作用。该激酶在Ser2、Thr54和Ser163等多个位点上磷酸化Tmod1。针对磷酸化位点的抗体将被用来识别细胞中修饰的Tmod。这些位点上的残基将被突变为Ala和Asp或Glu，从而相应地产生一个不可磷酸化的蛋白和一个拟磷化蛋白。这些突变对Tmod1功能的影响将在TM结合、雾化蛋白结合和肌动蛋白封顶实验中进行分析。Tmod修饰对肌原纤维形成的影响将在心肌细胞中进行检查。调节Tmod封帽活性的因素对于正确的肌原纤维形成是非常重要的。确定监管因素，无论它们是已知的还是未知的实体，将为我们理解对尖端及其上游监管的控制提供一个“缺失的环节”。

DESCRIPTION (provided by applicant): Regulation of the dynamics at actin's ends is of central importance in the assembly of thin filaments in the striated muscle sarcomeres, as well as in smooth muscle and non-muscle cells. An actin filament has two distinct ends: a fast-growing barbed end and a slower growing pointed end. Tropomodulin (Tmod) is a tropomyosin (TM) binding protein that binds to and regulates the dynamics of the pointed end. This proposal focuses on defining structure-function relationships in Tmod, and proteins that bind to and regulate its function. Of the many possible mechanisms that might influence dynamics at the actin pointed end, three that appear to be the most likely will be explored: isoform dependence in Tmod/TM interaction (Aim 1), identification of still unknown Tmod binding partners that change Tmod functional abilities (Aim 2) and Tmod phosphorylation (Aim 3). Actin capping is tight in in vitro experiments; yet, in living myocytes capping is transient. There must be a mechanism to regulate Tmod, either a protein that regulates by binding Tmod, or covalent modification. In Aim 1, model peptides of the N-terminus of TM that bind to Tmod, and of the two defined TM binding sites on Tmod will be used to learn the structural basis of the specificity of binding of different TMs to different Tmods. Binding will be assayed using native gel electrophoresis, cross-linking, and circular dichroism spectroscopy. The specificities will then be evaluated in pointed end elongation assays and in rescue experiments using Tmod1 null embryonic stem cells. Aim 2 tests the hypothesis: The Leucine-Rich- Repeat (LRR) domain in Tmod binds factors that regulate its function. Impure actin preparations from skeletal muscle contain a factor that negatively regulates Tmod capping. The activity depends on the presence of the LRR domain, a motif typically involved in protein-protein interactions. The aim will be to purify, identify and characterize the regulatory factor. In Aim 3 phosphorylation of Tmod by TRPM7 kinase will be studied. The kinase phosphorylates Tmod1 in multiple sites, Ser2, Thr54 and Ser163. Antibodies against phosphorylated sites will be used to identify modified Tmod in cells. Residues in the sites will be mutated to Ala and Asp or Glu to create an unphosphorylatable and a phosphomimetic proteins, correspondingly. The influence of these mutations on Tmod1 function will be assayed in TM-binding, nebulin-binding and actin-capping experiments. The effects of Tmod modifications on myofibrillogenesis will be checked in myocytes. Factors regulating Tmod capping activity are of great importance in proper myofibril formation. Determining the regulatory factors, whether they are a known or unknown entity, will provide a "missing link" in our understanding of control of the pointed end, and its upstream regulation.