REGULATION OF ACTIN CAPPING PROTEIN

肌动蛋白加帽蛋白的调节

基本信息

批准号：
8464156
负责人：
JOHN A COOPER
金额：
$ 27.87万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-05-01 至 2016-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8464156
关键词：
Abbreviations Actins Address Affect Agaricales Amino Acid Motifs Binding Binding Proteins Binding Sites Biochemical Biological Assay C-terminal CD2-associated protein Cell physiology Cells Complex Cytoskeleton Defect Development Filament Genes Goals Immigration In Vitro Inflammatory Integrins Leucine-Rich Repeat Life Lipids Location Membrane Membrane Proteins Microfilaments Microscopy Modeling Molecular Molecular Conformation Movement Myosin ATPase Myosin Type I Phenotype Physiological Plus End of the Actin Filament Process Protein Binding Domain Protein Isoforms Proteins RNA Cap-Binding Proteins Recruitment Activity Regulation Research Role Shapes Site Structure Subcellular structure Surface Testing Vertebrates Work actin capping protein base cancer cell cell motility human disease inhibitor/antagonist mutant novel prevent response

项目摘要

Our goals are to elucidate the molecular mechanisms that control the assembly and disassembly of actin filaments in cells and to understand how actin assembly dynamics contribute to cell movement. The creation of free barbed ends of actin filaments is a critical determinant of actin assembly, and capping those ends is necessary to produce force and movement at membranes. Here, we investigate the function of the heterodimeric barbed-end capping protein (CP) and a set of membrane-associated proteins that contain a conserved CP-binding motif, called CPI, but are otherwise unrelated. CARMIL, which contains the CPI motif and a second CP-binding motif, called CSI (CARMIL- specific interacting), is a potent inhibitor of CP with the ability to create free barbed ends by uncapping capped filaments. Uncapping is important because the turnover rates of CP and actin filaments in cells are faster by orders of magnitude than those observed with purified proteins in vitro. Other CPI-motif proteins, including CD2AP, Cin85, CKIP-1, WASHCAP(FAM21) and CapZIP bind CP but inhibit less well, suggesting that they may target active CP to membrane compartments. Aim 1: How Do Regulators of Capping Protein Work? To understand how CP regulation works, we defined the actin-binding sites on CP, and we produced crystal structures for CP in complex with CP- binding proteins. CPI motifs bind to a common site on CP at a distance from the actin-binding sites. CARMIL binding causes an allosteric change in the conformation of the actin-binding sites. Now, we ask whether the cellular function of the various CPI-motif proteins is to inhibit CP or to recruit active CP to a location in the cell. In vitro, we will compare the abilities of the proteins to bind and inhibit CP in biochemical assays with purified components. In cells, we will determine how targeting and incorporation of CP into the actin cytoskeleton depends on the CP-interacting proteins and how their interaction with CP affects local actin assembly and movement. Aim 2: How Does CARMIL1 Function in Cells? CARMIL is important for cell migration and other actin- related functions in metazoan cells. Vertebrates express three conserved CARMIL genes. We discovered that CARMIL1 and CARMIL2 are both important for cell migration and that they have distinct non-overlapping functions in a single migrating cell. CARMIL1 interacts with the dual-GEF Trio, activates Rac1, and stimulates lamellipodia formation. We plan to investigate the molecular basis of these phenotypes and interactions by a combination of localization, biochemical, knockdown, and expression approaches. COOPER, John A. 1R01 GM095509-01A1

我们的目标是阐明控制组装和组装的分子机制细胞中肌动蛋白丝的分解并了解肌动蛋白组装动力学如何有助于细胞运动。肌动蛋白丝自由倒刺末端的产生是肌动蛋白的关键决定因素组装，并盖住这些末端对于在膜上产生力和运动是必要的。这里，我们研究了异二聚体倒刺封端蛋白（CP）的功能和一组膜相关蛋白，含有保守的 CP 结合基序，称为 CPI，但否则无关。 CARMIL，包含 CPI 基序和第二个 CP 结合基序，称为 CSI (CARMIL- 特异性相互作用），是一种有效的 CP 抑制剂，能够通过开盖产生自由的倒刺末端加盖细丝。脱帽很重要，因为细胞中 CP 和肌动蛋白丝的周转率比体外纯化蛋白质观察到的速度快几个数量级。其他 CPI 主题蛋白质，包括 CD2AP、Cin85、CKIP-1、WASHCAP(FAM21) 和 CapZIP 结合 CP，但抑制较少好吧，这表明他们可能将活性 CP 靶向膜室。目标 1：封盖蛋白调节剂如何发挥作用？为了了解 CP 监管如何运作，我们定义了 CP 上的肌动蛋白结合位点，我们生成了 CP 与 CP- 复合物的晶体结构结合蛋白。 CPI 基序与 CP 上距离肌动蛋白结合位点一定距离的共同位点结合。 CARMIL 结合导致肌动蛋白结合位点构象发生变构变化。现在，我们询问各种 CPI 基序蛋白的细胞功能是抑制 CP 还是招募活性 CP 到单元格中的某个位置。在体外，我们将比较蛋白质结合和抑制 CP 的能力使用纯化成分进行生化测定。在细胞中，我们将确定如何靶向和 CP 掺入肌动蛋白细胞骨架取决于 CP 相互作用蛋白及其如何与 CP 的相互作用影响局部肌动蛋白的组装和运动。目标 2：CARMIL1 在细胞中如何发挥作用？ CARMIL 对于细胞迁移和其他肌动蛋白非常重要后生动物细胞中的相关功能。脊椎动物表达三个保守的 CARMIL 基因。我们发现 CARMIL1 和 CARMIL2 对细胞迁移都很重要，并且它们具有单个迁移细胞中具有不同的非重叠功能。 CARMIL1 与双 GEF Trio 相互作用，激活 Rac1，并刺激片状伪足的形成。我们计划研究其分子基础这些表型和相互作用通过定位、生化、敲低和表达方式接近。库珀，约翰 A. 1R01 GM095509-01A1