Molecular Mechanisms of Actin Cytoskeleton Dynamics

肌动蛋白细胞骨架动力学的分子机制

• 批准号: 8979897
• 负责人: JIANPENG MA
• 金额: $ 34.08万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8979897
• 关键词: ATP Hydrolysis; Actin-Binding Protein; Actins; Adenylyl Imidodiphosphate; Adopted; Binding; Binding Sites; Biological Process; Cell physiology; Cells; Communicable Diseases; Complex; Computer Simulation; Crystallization; Cytokinesis; Cytoskeleton; Development; Dimerization; Disease; Elongation Factor; F-actin-binding proteins; Family; Filament; Goals; Hereditary Disease; Homo; Infection; Investigation; Mediating; Molecular; Molecular Conformation; Morphogenesis; Muscle Development; Muscular Dystrophies; Mutation; Myomatous neoplasm; Neoplasm Metastasis; Neurodegenerative Disorders; Process; Protein Fragment; Proteins; Refractory; Research; Role; Staging; Structure; Surface; Testing; Tropomyosin; base; cell motility; design; drug development; human disease; innovation; member; monomer; mutant; neurogenesis; novel; prevent; profilin; public health relevance; research study; success; tumor initiation; tumorigenesis

 DESCRIPTION (provided by applicant): The inability to obtain atomic structures on actin cytoskeleton has severely hindered our understanding of this most abundant eukaryotic protein and its dynamic turnovers in performing a myriad of cellular functions such as cell motility, cytokinesis and morphogenesis through interactions with hundreds of actin-binding proteins. This application describes an innovative double-mutant strategy to overcome this longstanding barrier in the actin cytoskeleton field. Indeed, this novel double-mutant strategy has enabled the PIs' successful determination of the very first atomic crystal structure of actin-nucleator complex (actin-Cobl complex). In addition, a similar approach has been used to solve the structure of actin with a bacterial effector, VopL. The observed non-filament-like conformation in actin-Cobl structure and filament-like conformation in actin-VopL structure together suggest that both types of conformation are fully accessible to an actin complex obtained via the double-mutant strategy, thus its true conformation is most likely preserved. The experiments proposed in this application will apply the double-mutant strategy to three most divergent members of the latest class actin nucleators characterized by the presence of tandem actin-binding sites. The goal is to decipher their molecular mechanisms of actin nucleation, the roles of ATP hydrolysis in their functional cycle, and how they collaborate with specific cellular components to fulfill their functions. This goal will be achieved in three Specific Aims using combined structural and functional approaches: Aim 1: Mechanisms of Cobl-mediated actin nucleation; Aim 2: Mechanistic study of actin nucleation by Lmod; and Aim 3: Mechanistic study of APC-mediated actin nucleation. The extensive preliminary studies presented in this application suggest a high promise of success for the proposed research. Results from this study will be significant not only in elucidating the molecular mechanisms of their respective roles in neurogenesis, muscle development and tumor initiation, but also in unraveling some long-sought-after general underpinnings for de novo actin nucleation, a process underlying every stage of mammalian development as well as many types of pathogenic infection. By providing a detailed atomic gallery of how de novo actin nucleation is accomplished and regulated, this study will stimulate deeper mechanistic investigations on these nucleators as well as discovery and characterization of new actin nucleators. More importantly, this application will validate the double-mutant strategy on proteins/protein fragments with and without actin nucleation activities, thus providing sufficient proof-of-principle for extending this approach to many other actin-involved biological processes beyond actin nucleation. Ultimately, the research along this line is expected to directly benefit the treatment of many forms of human diseases due to actin cytoskeleton malfunctions including neurodegenerative disorders, muscular dystrophy, tumorigenesis and metastasis.

 描述(申请人提供)：无法获得肌动蛋白细胞骨架上的原子结构严重阻碍了我们对这一最丰富的真核蛋白质及其通过与数百种肌动蛋白结合蛋白相互作用而执行多种细胞功能(如细胞运动、胞质分裂和形态形成)的动态转换的理解。本申请描述了一种创新的双突变策略，以克服肌动蛋白细胞骨架领域的这一长期障碍。事实上，这种新的双突变策略已经使PI成功地确定了肌动蛋白-核仁复合体的第一个原子晶体结构 (肌动蛋白-眼镜蛇复合体)。此外，一种类似的方法也被用来解决带有细菌效应器VopL的肌动蛋白的结构。在肌动蛋白-COBL结构中观察到的非丝状构象和在肌动蛋白-VopL结构中观察到的丝状构象共同表明，这两种构象都可以完全被通过双突变策略获得的肌动蛋白复合体所结合，因此它的真实构象很有可能被保留下来。本申请中提出的实验将把双突变策略应用于最新一类肌动蛋白核仁的三个最具分歧的成员，其特征是存在串联的肌动蛋白结合位点。我们的目标是破译它们的肌动蛋白成核的分子机制，ATP水解在它们的功能周期中的作用，以及它们如何与特定的细胞成分合作来完成它们的功能。这一目标将通过结构和功能相结合的方法在三个具体目标中实现：目标1：COBL介导的肌动蛋白成核机制；目标2：LMOD对肌动蛋白成核的机制研究；以及目标3：APC介导的肌动蛋白成核的机制研究。本申请中提出的广泛的初步研究表明，拟议的研究有很高的成功前景。这项研究的结果不仅对于阐明它们在神经发生、肌肉发育和肿瘤起始中各自作用的分子机制具有重要意义，而且对于解开一些长期寻求的从头肌动蛋白成核的一般基础也具有重要意义，从头肌动蛋白成核是哺乳动物发育的每个阶段以及许多类型病原体感染的潜在过程。通过提供一个关于从头肌动蛋白成核是如何完成和调控的详细的原子图库，这项研究将促进对这些核子的更深层次的机制研究，以及对新的肌动蛋白核子的发现和表征。更重要的是，这一应用将验证具有和不具有肌动蛋白核活性的蛋白质/蛋白质片段上的双突变策略，从而提供 将这一方法扩展到肌动蛋白成核以外的许多其他涉及肌动蛋白的生物过程的充分原则证明。最终，这一方向的研究有望直接受益于多种形式的人类疾病的治疗，这些疾病是由肌动蛋白细胞骨架故障引起的，包括神经退行性疾病、肌肉营养不良、肿瘤发生和转移。