Structural-Functional Basis of Actin Cytoskeleton Dynamics

肌动蛋白细胞骨架动力学的结构功能基础

• 批准号: 10116413
• 负责人: ROBERTO DOMINGUEZ
• 金额: $ 36.04万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10116413
• 关键词: ATP phosphohydrolase; Acetylation; Acetyltransferase; Actins; Address; Architecture; Area; Baculoviruses; Binding; Binding Sites; Biochemical; Biology; Case Study; Cell division; Cell physiology; Cells; Collaborations; Complex; Crystallization; Cytoskeleton; Data; Development; Diagnosis; Disease; Elongation Factor; Environment; Family; Filament; Functional disorder; Goals; Grant; Health; Immune response; In Vitro; Knowledge; Lead; Mammalian Cell; Microfilaments; Modification; Molecular; Morphology; Myosin ATPase; N-terminal; Nemaline Myopathies; Neoplasm Metastasis; Neurodegenerative Disorders; Nonmuscle Myosin Type IIA; Norway; Organelles; Pathway interactions; Pilot Projects; Post-Translational Protein Processing; Process; Protein Acetylation; Protein Isoforms; Proteins; Publishing; Reaction; Reporting; Research; Role; Series; Source; Structure; System; Testing; Time; Tropomyosin; Variant; Work; base; cell motility; human disease; innovation; interdisciplinary approach; interest; mutant; preference; profilin; response; scaffold; scale up; skeletal; tool; trait; treatment strategy; tropomodulin; ATP phosphohydrolase; Acetylation; Acetyltransferase; Actins; Address; Architecture; Area; Baculoviruses; Binding; Binding Sites; Biochemical; Biology; Case Study; Cell division; Cell physiology; Cells; Collaborations; Complex; Crystallization; Cytoskeleton; Data; Development; Diagnosis; Disease; Elongation Factor; Environment; Family; Filament; Functional disorder; Goals; Grant; Health; Immune response; In Vitro; Knowledge; Lead; Mammalian Cell; Microfilaments; Modification; Molecular; Morphology; Myosin ATPase; N-terminal; Nemaline Myopathies; Neoplasm Metastasis; Neurodegenerative Disorders; Nonmuscle Myosin Type IIA; Norway; Organelles; Pathway interactions; Pilot Projects; Post-Translational Protein Processing; Process; Protein Acetylation; Protein Isoforms; Proteins; Publishing; Reaction; Reporting; Research; Role; Series; Source; Structure; System; Testing; Time; Tropomyosin; Variant; Work; base; cell motility; human disease; innovation; interdisciplinary approach; interest; mutant; preference; profilin; response; scaffold; scale up; skeletal; tool; trait; treatment strategy; tropomodulin

Project Summary: The long-term goal is to understand the structural and functional mechanisms that control actin cytoskeleton dynamics in health and disease. In its fourth cycle, this grant addresses important gaps of knowledge in previous focus areas, while also expanding into new areas in response to recent developments in the field. Building upon the recent discovery by this lab in collaboration with a group in Norway of actin's dedicated N- terminal acetyltransferase (Naa80), Aim-1 will focus on the mechanism and function of actin N-terminal acetylation (N-acetylation) and isoform variations. Aim-1a will determine the mechanism of N-acetylation, including biochemical and cellular studies of the Naa80-actin interaction and a series of crystal structures of intermediate reaction steps, and using different actin isoforms. Aim-1b will test the hypothesis that myosin activity is strongly dictated by actin isoform variations and N-acetylation. This will resolve an enduring deficiency in the field; most myosin studies have used α-skeletal actin, overlooking the fact that differences among actin isoforms concentrate at the N-terminus, which is also acetylated and forms part of the myosin- binding site. Aim-1c will explore the role of actin N-acetylation and profilin/actin isoform variations on formin function. Formins are the most important actin filament elongation factors in cells, and this group has found that actin N-acetylation has a profound effect on filament elongation. It is further postulated that actin isoform variations that concentrate at the N-terminus will have an equally strong effect on formin activity. Aim-2 proposes new strategies to tackle persisting questions in the area of actin nucleation, which constitutes a long-standing interest of this lab. Aim-2a will study in parallel Tmods and Lmods, which despite having a common fold have evolved different activities – pointed-end capping and nucleation, respectively. The source of the functional differences between these two subfamilies will be studied with a focus on Lmod3, implicated in nemaline myopathy. An innovative strategy is proposed to determine the structure of Tmod at the pointed end. It is finally investigated, in vitro and in cells, how specific Tmod and Tropomyosin isoforms interact with each other to assemble morphologically and functionally distinct actin networks. Aim-2b will tackle the long- standing problem of how WASP-family Nucleation Promoting Factors interact with and activate branch formation by the Arp2/3 complex. The plans build upon the ability to perform biochemical and structural studies on the baculovirus-expressed Arp2/3 complex and subcomplexes. Extensive preliminary and published work provide the scientific premise and support feasibility.

项目摘要： 长期目标是了解控制肌动蛋白细胞骨架的结构和功能机制 健康和疾病的动力。在其第四个周期中，该赠款解决了重要的知识差距 以前的重点领域，同时还可以响应该领域的最新发展而扩展到新领域。 基于该实验室最近发现的基础，与挪威的一个团队合作， 末端乙酰转移酶（NAA80），AIM-1将集中于肌动蛋白N末端的机理和功能 乙酰化（N-乙酰化）和同工型变化。 AIM-1A将确定N-乙酰化的机制， 包括NaA80-肌动蛋白相互作用的生化和细胞研究以及一系列的晶体结构 中间反应步骤，并使用不同的肌动蛋白同工型。 AIM-1B将检验肌球蛋白的假设 活性由肌动蛋白同工型变异和N-乙酰化决定。这将解决一个持久的 该领域的不足；大多数肌球蛋白研究都使用了α-骨骼肌动蛋白，忽视了差异 在肌动蛋白同工型中，浓缩的N-末端也是乙酰化的，并构成了肌球蛋白的一部分 绑定位点。 AIM-1C将探索肌动蛋白N-乙酰化和profilin/肌动蛋白同工型的作用 Formin功能。造型是细胞中最重要的肌动蛋白丝脱纤维剂，该组具有 发现肌动蛋白N-乙酰化对细丝伸长有深远的影响。进一步发布了肌动蛋白 集中在N末端的同工型变异对造型活性具有同样强大的影响。 AIM-2提案采取新的策略，以解决肌动蛋白成核领域的持续问题，该策略构成一个 这个实验室的长期兴趣。 AIM-2A将以并行的TMOD和LMOD进行研究，这些愿望具有 共同的折叠已经演变出不同的活动 - 分别尖端的封盖和成核。来源 这两个亚家族之间的功能差异将被研究，重点是LMOD3 在Nemaline肌病中。提出了一种创新的策略来确定尖头的TMOD结构 结尾。它最终在体外和细胞中研究了特定的Tmod和Tropomyosin同工型如何与 彼此以形态和功能上不同的肌动蛋白网络组装。 AIM-2B将解决长期 黄蜂家庭成核如何促进因子与分支相互作用并激活分支的站立问题 ARP2/3复合物的格式。该计划以执行生化和结构的能力为基础 对杆状病毒表达的ARP2/3复合物和子复合物的研究。广泛的初步和 已发表的工作提供了科学的前提和支持可行性。