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

• 批准号: 8399736
• 负责人: Alla S. Kostyukova
• 金额: $ 36万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8399736
• 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; Health; 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 Cells; Muscle Contraction; 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 Myocytes; Specificity; Striated Muscles; Structure; Structure-Activity Relationship; Testing; Thin Filament; Tissues; Tropomyosin; alanylaspartic acid; alpha helix; base; cell motility; cell type; crosslink; embryonic stem cell; gel electrophoresis; nebulin; overexpression; pointed protein; protein protein interaction; research study; tropomodulin

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.

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细胞中细丝的组装中具有重要意义。肌动蛋白丝有两个不同的末端：一个快速生长的倒刺末端和一个缓慢生长的尖头末端。原肌球蛋白（Tmod）是一种原肌球蛋白（TM）结合蛋白，结合并调节尖端的动力学。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，有三种似乎是最有可能的将被探索：Tmod/TM相互作用中的同种型依赖性（目的1），识别改变Tmod功能能力的未知Tmod结合伴侣（目的2）和Tmod磷酸化（目的3）。在体外实验中，肌动蛋白帽是紧密的;然而，在活的肌细胞中，帽是短暂的。必须有一种调节Tmod的机制，要么是通过结合Tmod进行调节的蛋白质，要么是共价修饰。在目的1中，结合Tmod的TM的N-末端的模型肽和Tmod上的两个限定的TM结合位点的模型肽将用于了解不同TM与不同Tmod的结合特异性的结构基础。将使用非变性凝胶电泳、交联和圆二色谱法测定结合。然后将在尖端延伸测定和使用Tmod 1无效胚胎干细胞的拯救实验中评价特异性。目的2检验Tmod中的亮氨酸丰富重复序列（LRR）结构域与调节其功能的因子结合的假设。来自骨骼肌的不纯肌动蛋白制剂含有负调节Tmod帽的因子。活性取决于LRR结构域的存在，LRR结构域是典型地参与蛋白质-蛋白质相互作用的基序。目的是纯化、鉴定和表征调节因子。在目标3中，将研究TRPM 7激酶对Tmod的磷酸化。该激酶在多个位点（Ser 2、Thr 54和Ser 163）磷酸化Tmod 1。针对磷酸化位点的抗体将用于鉴定细胞中修饰的Tmod。位点中的残基将突变为Ala和Asp或Glu以相应地产生不可磷酸化的蛋白质和磷酸模拟蛋白质。这些突变对Tmod 1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修饰对肌原纤维发生的影响。调节Tmod加帽活性的因子在适当的肌原纤维形成中非常重要。确定调节因素，无论它们是已知的还是未知的实体，将为我们理解尖端控制及其上游调节提供“缺失的环节”。

项目成果

Tropomodulins and tropomyosins: working as a team.

• DOI: 10.1007/s10974-013-9349-6
• 发表时间: 2013-08
• 期刊: Journal of muscle research and cell motility
• 影响因子: 2.7
• 作者: Colpan M;Moroz NA;Kostyukova AS
• 通讯作者: Kostyukova AS

Mutations changing tropomodulin affinity for tropomyosin alter neurite formation and extension.

改变原调节蛋白对原肌球蛋白亲和力的突变改变了神经突的形成和延伸。

• DOI: 10.7717/peerj.7
• 发表时间: 2013
• 期刊: PeerJ
• 影响因子: 2.7
• 作者: Moroz,Natalia;Guillaud,Laurent;Desai,Brinda;Kostyukova,AllaS
• 通讯作者: Kostyukova,AllaS

Systematic analysis of tropomodulin/tropomyosin interactions uncovers fine-tuned binding specificity of intrinsically disordered proteins.

原调节蛋白/原肌球蛋白相互作用的系统分析揭示了本质上无序蛋白质的微调结合特异性。

• DOI: 10.1002/jmr.1093
• 发表时间: 2011
• 期刊: Journal of molecular recognition : JMR
• 作者: Uversky,VladimirN;Shah,SamarP;Gritsyna,Yulia;Hitchcock-DeGregori,SarahE;Kostyukova,AllaS
• 通讯作者: Kostyukova,AllaS

Binding of human angiogenin inhibits actin polymerization.

• DOI: 10.1016/j.abb.2009.12.024
• 发表时间: 2010-03-01
• 期刊: Archives of biochemistry and biophysics
• 影响因子: 3.9
• 作者: Pyatibratov MG;Tolkatchev D;Plamondon J;Xu P;Ni F;Kostyukova AS
• 通讯作者: Kostyukova AS

New insights into the role of angiogenin in actin polymerization.

• DOI: 10.1016/b978-0-12-394306-4.00011-3
• 发表时间: 2012
• 期刊: International review of cell and molecular biology
• 作者: Pyatibratov MG;Kostyukova AS
• 通讯作者: Kostyukova AS