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

描述（由申请人提供）：肌动蛋白末端的动力学调节在横纹肌肌节以及平滑肌和非肌肉细胞中细丝的组装中至关重要。肌动蛋白丝有两个不同的末端：一个快速生长的倒钩端和一个生长较慢的尖端。原调蛋白（Tropomodulin, Tmod）是一种原肌球蛋白（tropomyosin， TM）结合蛋白，与尖端结合并调节其动力学。本研究的重点是定义Tmod的结构-功能关系，以及结合和调节其功能的蛋白质。在可能影响肌动蛋白尖端动力学的许多可能机制中，有三个似乎是最有可能的将被探索：Tmod/TM相互作用中的异构体依赖性（Aim 1），识别仍然未知的改变Tmod功能能力的Tmod结合伙伴（Aim 2）和Tmod磷酸化（Aim 3）。体外实验肌动蛋白封盖紧密；然而，在活的肌细胞中，封盖是短暂的。一定存在一种调节Tmod的机制，要么是一种通过结合Tmod来调节的蛋白质，要么是共价修饰。在Aim 1中，我们将使用TM与Tmod结合的n端模型肽，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结合特异性的结构基础。结合将分析使用天然凝胶电泳，交联，和圆二色光谱。然后将在尖端延伸试验和使用Tmod1空胚胎干细胞的抢救实验中评估特异性。目的2验证假设：Tmod中的Leucine-Rich- Repeat （LRR）结构域结合调节其功能的因子。来自骨骼肌的不纯肌动蛋白制剂含有负调节Tmod封顶的因子。这种活性取决于LRR结构域的存在，LRR结构域是一种典型的参与蛋白质-蛋白质相互作用的基序。目的是纯化、鉴定和表征调节因子。在Aim 3中，将研究TRPM7激酶对Tmod的磷酸化作用。该激酶磷酸化Tmod1的多个位点，包括Ser2、Thr54和Ser163。针对磷酸化位点的抗体将用于鉴定细胞中修饰的Tmod。这些位点上的残基将突变为Ala和Asp或Glu，从而相应地产生不可磷酸化和拟磷蛋白。这些突变对Tmod1功能的影响将在tm结合、nebulin结合和actin-capping实验中进行分析。Tmod修饰对肌原纤维形成的影响将在肌细胞中进行检查。调节Tmod帽盖活性的因子对肌原纤维的形成非常重要。确定监管因素，无论它们是已知的还是未知的实体，将为我们对尖端控制及其上游监管的理解提供一个“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.