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

影响肌动蛋白丝尖端动力学调节的因素

基本信息

批准号：
7762195
负责人：
Alla S. Kostyukova
金额：
$ 29.34万
依托单位：
UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-02-01 至 2013-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7762195
关键词：
Actins Activities of Daily Living Affinity Antibodies Binding Binding Proteins Binding Sites Biological Assay C-terminal Cells Cellular Morphology Circular Dichroism Spectroscopy Complex Congenital Heart Defects Cytoskeleton Dependence Dilated Cardiomyopathy Disease Embryo Gelsolin Goals Heart In Vitro Knockout Mice Lead Learning Length Leucine-Rich Repeat Life Link Location Microfilaments Minus End of the Actin Filament Modeling Modification Molecular Mus Muscle Muscle Cells Mutate Mutation Myofibrillogenesis Myofibrils N-terminal Peptides Phosphorylation Phosphorylation Site Phosphotransferases Play Preparation Process Protein Binding Protein Isoforms Proteins Regulation Role Sarcomeres Site Skeletal Muscle Smooth Muscle Specificity Striated Muscles Structure Structure-Activity Relationship Testing Thin Filament Thromboplastin Tissues Tropomyosin alanylaspartic acid alpha helix base cell motility cell type crosslink embryonic stem cell gel electrophoresis nebulin overexpression pointed protein protein protein interaction public health relevance research study tropomodulin

项目摘要

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. PUBLIC HEALTH RELEVANCE: Tropomodulin is a tropomyosin-binding protein which caps the slow-growing (pointed) end of the actin filament and therefore regulates its dynamics. Its overexpression in the heart of mice leads to myofibril degeneration and dilated cardiomyopathy. In a tropomodulin-null mouse, heart defects lead to embryonic lethality. Factors regulating Tmod function are of great importance in proper myofibril formation. The information obtained in this study will help us to understand the formation of the capping complex at the pointed end and its function in regulating dynamics of the actin filament in cells and tissues.

描述（由申请人提供）：肌动蛋白末端动力学的调节对于横纹肌肌节以及平滑肌和非肌肉细胞中细丝的组装至关重要。肌动蛋白丝有两个不同的末端：一个快速生长的带刺末端和一个生长较慢的尖端。原调节蛋白 (Tmod) 是一种原肌球蛋白 (TM) 结合蛋白，可结合并调节尖端的动态。该提案的重点是定义 Tmod 中的结构-功能关系，以及结合并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，将探讨最有可能的三种机制：Tmod/TM 相互作用中的异构体依赖性（目标 1）、识别仍未知的改变 Tmod 功能的 Tmod 结合伙伴能力（目标 2）和 Tmod 磷酸化（目标 3）。体外实验中肌动蛋白封盖紧密；然而，在活的心肌细胞中，加帽是短暂的。一定有一种机制来调节Tmod，要么是通过结合Tmod来调节的蛋白质，要么是共价修饰。在目标 1 中，与 Tmod 结合的 TM N 末端的模型肽以及 Tmod 上两个定义的 TM 结合位点的模型肽将用于了解不同 TM 与不同 Tmod 结合的特异性的结构基础。将使用天然凝胶电泳、交联和圆二色光谱法来测定结合。然后，将在尖端伸长测定和使用 Tmod1 无效胚胎干细胞的救援实验中评估特异性。目标 2 检验假设：Tmod 中的富含亮氨酸重复序列 (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。该活性取决于 LRR 结构域的存在，该结构域通常涉及蛋白质-蛋白质相互作用。目的是纯化、识别和表征监管因素。在目标 3 中，将研究 TRPM7 激酶对 Tmod 的磷酸化。该激酶在多个位点（Ser2、Thr54 和 Ser163）磷酸化 Tmod1。针对磷酸化位点的抗体将用于识别细胞中修饰的 Tmod。位点中的残基将突变为丙氨酸和天冬氨酸或谷氨酸，以相应地产生不可磷酸化的和拟磷酸化的蛋白质。这些突变对 Tmod1 功能的影响将在 TM 结合、星云蛋白结合和肌动蛋白加帽实验中进行测定。将在肌细胞中检查 Tmod 修饰对肌原纤维生成的影响。调节 Tmod 封端活性的因素对于适当的肌原纤维形成非常重要。确定监管因素，无论它们是已知的还是未知的实体，都将为我们理解尖端控制及其上游监管提供一个“缺失的环节”。公共健康相关性：原调节蛋白是一种原肌球蛋白结合蛋白，它覆盖肌动蛋白丝缓慢生长（尖的）末端，从而调节其动态。它在小鼠心脏中的过度表达会导致肌原纤维变性和扩张型心肌病。在原调节蛋白缺失的小鼠中，心脏缺陷会导致胚胎死亡。调节 Tmod 功能的因素对于正常肌原纤维的形成非常重要。本研究获得的信息将帮助我们了解尖端加帽复合物的形成及其在调节细胞和组织中肌动蛋白丝动力学中的功能。