Allosteric Regulation of Actin Capping Protein: Mechanism and Significance

肌动蛋白加帽蛋白的变构调节：机制和意义

• 批准号: 10330809
• 负责人: JOHN A COOPER
• 金额: $ 55.24万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-19 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10330809
• 关键词: Actins; Allosteric Regulation; Amino Acid Motifs; Arthritis; Binding; Biochemical; Biological Assay; Biological Phenomena; Biophysical Process; Cell Shape; Cell physiology; Cells; Cellular Membrane; Chimera organism; Diffuse; Disease; Dissociation; Endothelial Cells; Family; Immune; Infection; Knowledge; Lipids; Malignant Neoplasms; Measurement; Membrane; Microfilaments; Molecular; Molecular Conformation; Molecular Genetics; Physiological; Physiological Processes; Plus End of the Actin Filament; Process; Proteins; Research; Shapes; Side; Site; Surface; System; Testing; Time; Work; actin capping protein; cancer cell; cell motility; migration; molecular dynamics; monolayer; movie; novel; polymerization; reconstitution; single-molecule FRET

Project Summary/Abstract Actin assembly underlies and drives many biological phenomena. Barbed ends of actin filaments, the major sites of polymerization, are controlled by the heterodimeric actin capping protein (CP). CP is regulated by the direct binding of CPI-motif proteins and the protein V-1. These two classes of regulators, CPI-motif proteins and V-1, bind to opposite sides of CP, and they induce conformational changes in CP that allosterically antagonize the binding of the other class. We are studying the molecular biophysical mechanism of these allosteric regulators. We are also studying the physiological function of CP and its regulators, using biochemical reconstitution with purified components, along with molecular genetic perturbations of living cells. Our biochemical studies will test a novel hypothesis for how CP regulators function in cells. Cells contain stoichiometric amounts of V-1 in micromolar concentrations, sufficient to inhibit nearly all of the cellular CP. V-1 is highly diffusible, and V-1 sterically blocks the ability of CP to cap actin filaments. CPI-motif proteins are targeted to membranes, and their CPI motifs allosterically induce the dissociation of V- 1, thus activating CP locally at the membrane. We are testing this hypothesis by determining the molecular biophysical mechanism of the allostery, and by testing the functions of the CPI-motif proteins with respect to cell motility and migration. We have discovered key differences in the biochemical activities of different families of CPI-motif proteins, and we are now using that information to investigate the allosteric mechanism, by combining single-molecule FRET measurements with molecular dynamics simulations. In addition, we are using purified proteins and lipids in a biochemical reconstitution system that induces actin assembly at a surface, thereby mimicking actin polymerization at cellular membranes. We are also using our discoveries about biochemical activities of CPI motifs to test cellular functions of CPI-motif proteins, using chimeras constructed from different CPI- motif proteins with molecular genetic perturbations and real-time movies of the motility phenomena of living cells. Our cell motility assays employ a system of endothelial cell monolayers with transmigrating immune and cancer cells, mimicking the physiological process of transendothelial migration.

项目概要/摘要 肌动蛋白组装是许多生物现象的基础和驱动因素。末端有倒刺 肌动蛋白丝的数量（聚合的主要位点）由异二聚体控制 肌动蛋白加帽蛋白（CP）。 CP 通过 CPI 基序蛋白的直接结合进行调节 和蛋白质V-1。这两类调节因子（CPI 基序蛋白和 V-1）结合 CP 的相对侧，并且它们引起 CP 的构象变化 变构拮抗另一类的结合。我们正在研究分子 这些变构调节剂的生物物理机制。我们也在研究 CP及其调节剂的生理功能，使用生化重建 纯化的成分，以及活细胞的分子遗传扰动。 我们的生化研究将检验 CP 调节剂如何发挥作用的新假设 在细胞中发挥作用。细胞含有微摩尔化学计量量的 V-1 浓度足以抑制几乎所有的细胞 CP。 V-1 具有高度扩散性， V-1 在空间上阻断 CP 封盖肌动蛋白丝的能力。 CPI 基序蛋白是 靶向膜，其 CPI 基序变构诱导 V-解离 1，从而在膜上局部激活CP。 我们正在通过确定分子生物物理学来检验这一假设 变构机制，并通过测试 CPI 基序蛋白的功能 关于细胞运动和迁移。我们发现了关键的差异 不同 CPI 基序蛋白家族的生化活性，我们现在正在使用 该信息通过结合单分子来研究变构机制 通过分子动力学模拟进行 FRET 测量。此外，我们正在使用 在诱导肌动蛋白的生化重建系统中纯化蛋白质和脂质 在表面组装，从而模拟细胞膜上的肌动蛋白聚合。 我们还利用我们关于 CPI 基序生化活性的发现来测试 CPI基序蛋白的细胞功能，使用由不同CPI基序构建的嵌合体 具有分子遗传扰动的基序蛋白和运动的实时电影 活细胞的现象。我们的细胞运动测定采用内皮细胞系统 具有迁移免疫细胞和癌细胞的单层细胞，模仿生理学 跨内皮迁移的过程。