REGULATION OF ACTIN CAPPING PROTEIN

肌动蛋白加帽蛋白的调节

基本信息

批准号：
8628846
负责人：
JOHN A COOPER
金额：
$ 28.88万
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/8628846
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 Cultured Cells Cytoskeleton Defect Development Filament Genes Goals Human 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 Zebrafish 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 in human cultured cells and zebrafish.

我们的目标是阐明控制组装的分子机制细胞中肌动蛋白丝的分解，并了解肌动蛋白如何组装动力学有助于细胞运动。肌动蛋白游离刺端的产生丝是肌动蛋白组装的关键决定因素，并且封盖这些末端是在膜上产生力和运动所必需的。在这里，我们调查异二聚体倒刺末端加帽蛋白（CP）和一组膜的功能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，能够产生通过解开加盖的细丝来释放倒刺末端。开封很重要，因为细胞中 CP 和肌动蛋白丝的周转率比细胞中快几个数量级体外纯化蛋白质观察到的结果。其他 CPI 基序蛋白，包括 CD2AP、Cin85、CKIP-1、WASHCAP(FAM21) 和 CapZIP 结合 CP，但抑制效果较差，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？了解 CP 调节方式工作原理，我们定义了 CP 上的肌动蛋白结合位点，并生成了晶体结构 CP 与 CP 结合蛋白形成复合物。 CPI 基序与 CP 上的一个共同位点结合距肌动蛋白结合位点的距离。 CARMIL 结合导致变构变化肌动蛋白结合位点的构象。现在，我们要问的是，细胞功能是否各种 CPI 基序蛋白的作用是抑制 CP 或将活性 CP 募集到细胞中的某个位置。在体外，我们将比较蛋白质在生化中结合和抑制 CP 的能力。用纯化的成分进行测定。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 相互作用蛋白以及它们与 CP 的相互作用如何影响局部肌动蛋白组装和运动。目标 2：CARMIL1 在细胞中如何发挥作用？ CARMIL 对细胞迁移很重要以及后生动物细胞中其他肌动蛋白相关的功能。脊椎动物表达三个保守的卡米尔基因。我们发现CARMIL1和CARMIL2对于细胞都很重要迁移，并且它们在单个迁移细胞中具有独特的非重叠功能。 CARMIL1 与双 GEF Trio 相互作用，激活 Rac1，并刺激片状伪足形成。我们计划研究这些表型的分子基础通过定位、生化、敲低和表达相结合的相互作用人类培养细胞和斑马鱼的方法。