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）：肌动蛋白末端的动力学调节在横纹肌肉肉瘤以及平滑肌和非肌肉细胞中的细丝组装中至关重要。肌动蛋白丝有两个不同的末端：一个快速生长的刺尾端和较慢的生长尖端。 Tropomodulin（TMOD）是一种结合并调节尖端的动力学的tropomyosin（TM）结合蛋白。该建议的重点是定义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指向端动力学的许多可能机制中，三种似乎最有可能的机制将被探索：TMOD/TM相互作用中的同工型依赖性（AIM 1），鉴定仍然未知的TMOD结合伙伴，这些伴侣会改变TMOD功能功能（AIM 2）和TMOD Phosphoration（AIM 3）。肌动蛋白限制在体外实验中很紧。然而，在活的心肌细胞中，封盖是短暂的。必须有一种调节TMOD的机制，即通过结合TMOD调节的蛋白质，或者是共价修饰。在AIM 1中，与TMOD结合的TM的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学习不同TMS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，交联和圆形二分法光谱法测定。然后，将在使用TMOD1 null胚胎干细胞中评估特异性的尖端伸长测定和救援实验。 AIM 2检验假设：TMOD中的富含亮氨酸重复（LRR）结构域结合调节其功能的因子。骨骼肌不纯净的肌动蛋白制剂包含一个负调节TMOD封盖的因素。该活性取决于LRR结构域的存在，LRR结构域通常参与蛋白质 - 蛋白质相互作用。目的是纯化，识别和表征调节因素。将研究TRPM7激酶对TMOD的AIM 3磷酸化。激酶在多个位点磷酸化TMOD1，SER2，THR54和SER163。针对磷酸化位点的抗体将用于鉴定细胞中的修饰TMOD。相应地，该位点中的残基将突变为ALA和ASP或GLU，以形成非磷酸化和磷酸化蛋白。这些突变对TMOD1功能的影响将在TM结合，云蛋白结合和肌动蛋白上限实验中分析。 TMOD修饰对肌原纤维发生的影响将在肌细胞中检查。调节TMOD上限活性的因素在适当的肌原纤维形成中非常重要。确定调节因素，无论是已知的还是未知的实体，都将在我们对尖端端的控制及其上游调节的理解中提供“缺失的联系”。

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